Purinostat mesylate for preventing and treating diffuse large b-cell lymphoma, analogue thereof, drug for combined use, and use

Abstract

Purinostat mesylate (PM) for preventing and treating diffuse large B-cell lymphoma (DLBCL), an analogue thereof, a drug for combined use, and the use. PM exhibits excellent in-vivo and in-vitro anti-tumor therapeutic effects on DLBCL (DEL and DHL) subtypes with poor prognosis, DLBCL subtypes having TP53 deletion and mutation combined with a plurality of poor prognosis gene mutations, and DLBCL having TP53 mutation with double expressors, which are superior to that of most existing DLBCL clinical therapy plans. A doublet or triplet therapy of PM with rituximab, R-CHOP, venetoclax and R-CHP also exhibits excellent in-vivo therapeutic effects against DLBCL and a plurality of subtypes. Thus, PM and the drug for combined use have an important clinical significance for the DLBCL and a plurality of subtypes.

Description

Prilostat mesylate and its analogs, combined drugs and uses for preventing and treating diffuse large B-cell lymphoma Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to prilocastat mesylate and its analogs, combined drugs and uses for preventing and treating diffuse large B-cell lymphoma. Background Art

[0002] Diffuse large B-cell lymphoma (DLBCL) is the most common pathological subtype of non-Hodgkin's lymphoma (NHL). The disease is highly heterogeneous in terms of clinical manifestations, biological characteristics and prognosis. According to the different cell origins, DLBCL is divided into three categories: germinal center B cell-like (GCB) subtype, activated B cell-like (ABC) subtype and unclassified type. In recent years, with the in-depth study of DLBCL and the improvement of detection technology, the genotyping of DLBCL has been updated, including MCD subtype (MYD88 L265P The MCD, N1, and A53 (ABC) subtypes have poor prognoses, with 5-year overall survival (OS) rates of only 40%, 27%, and 33%, respectively.

[0003] Advances in genotyping have advanced personalized treatment for DLBCL. However, double-hit lymphoma (DHL) or triple-hit lymphoma (THL) with ectopic expression of MYC and B-cell leukemia / lymphoma-2 (BCL-2) and / or B-cell leukemia / lymphoma-6 (BCL-6), double-expressing lymphoma (DEL) with overexpression of Myc and BCL-2 proteins, DLBCL patients with poor prognostic molecular biological features or gene mutations, and relapsed / refractory (R / R) DLBCL are more challenging to treat, representing an unmet clinical need and a current research focus.

[0004] In recent years, rapid progress has been made in the treatment of DLBCL, including preclinical research and clinical trials, with the emergence of new innovative drugs and therapies such as small molecule targeted drugs, bispecific antibodies, antibody-drug conjugates, and chimeric antigen receptor T cells (CAR-T). Among these, epigenetic regulatory drugs, histone deacetylase (HDAC) inhibitors, have become a hot research and development topic. Reference 1 (Locke FL, et al. Long-term safety and activity of axicabtagene ciloleucel in refractory large B-cell lymphoma (ZUMA-1): a single-arm, multicentre, phase 1-2 trial. The Lancet Oncology 2019, 20:31-42.) reports that 18 HDACs have been identified, which can be divided into four categories based on their homology to yeast proteins. Among them, HDAC class I includes HDAC1, HADC2, HDAC3, and HDAC8; HDAC class II is divided into HDAC class IIa (HDAC4, HADC5, HDAC7, and HDAC9) and HDAC class IIb (HDAC6 and HADC10); HDAC 11 belongs to HDAC class IV; all of the above are zinc-dependent HDACs, while HDAC class III is a class of NAD +Dependent proteins. Literature 2 (Liebers N, et al.Polatuzumab vedotin as a salvage and bridging treatment in relapsed or refractory large B-cell lymphomas. Blood advances 2021, 5:2707-16.) reports that there are currently five zinc-dependent HDAC inhibitors approved for clinical use by the US or China Food and Drug Administration. Panobinostat is used in combination to treat multiple myeloma, as well as vorinostat, romidepsin, belinostat and cedabendine, with the main indications being T-cell lymphoma. Among them, cedabendine selectively inhibits class I HDAC1, HDAC2, HDAC3 and class IIb HDAC10 subtypes, romidepsin selectively inhibits HDAC1 and HDAC2, and the other three are pan-HDAC inhibitors. Document 3-5 (Document 3: Awan FT et al. CD19 targeting of chronic lymphocytic leukemia with a novel Fc-domain-engineered monoclonal antibody. Blood 2010,115:1204-13. Document 4: Horton HM, et al. Potent in vitro and in vivo activity of an Fc-engineered anti-CD19monoclonal antibody against lymphoma and leukemia. Cancer research 2008,68:8049-57. Document 5: Salles G et al. Tafasitamab plus lenalidomide in relapsed or refractory diffuse large B-cell lymphoma (L-MIND): a multicentre, prospective, single-arm, phase 2 study. The Lancet Oncology 2020, 21: 978-88.) Studies have shown that HDAC Class I and IIb are more closely related to tumors, while the expression of class IIa and IV HDAC enzymes in the body is more closely related to cardiac, muscle, and immune regulation functions. Therefore, due to their inhibitory effects on class IIa and IV HDAC enzymes, pan-HDAC inhibitors can lead to more toxic side effects, including immunosuppression.Most existing HDAC inhibitors are pan-inhibitors, and their activity needs to be improved. Furthermore, clinical trials of multiple HDAC inhibitors for the treatment of DLBCL are underway, but the efficacy of single-agent treatment is suboptimal. This is especially true for patients with DHL, THL, or DEL in DLBCL, as well as those with molecular biological features or gene mutations associated with poor prognosis and R / R DLBCL, who experience extremely poor treatment responses and prognosis.

[0005] Therefore, there is an urgent need to find or develop new innovative drugs or therapies to treat DLBCL, especially DHL or THL, DEL and DLBCL patients with molecular biological characteristics or gene mutations associated with poor prognosis, and R / R DLBCL patients, and to improve their prognosis. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the present invention provides the use of prilocastat mesylate (PM), a selective inhibitor of histone deacetylase (HDAC) I and IIb, and its analogs in the preparation of drugs for treating diffuse large B-cell lymphoma (DLBCL), especially for patients with double hit lymphoma (DHL) or triple hit lymphoma (THL), double-expressing lymphoma (DEL), DLBCL patients with molecular biological characteristics or gene mutations associated with poor prognosis, and relapsed / refractory (R / R) DLBCL patients.

[0007] Definitions of Terms: PM: Purinostat mesylate; DLBCL: Diffuse large B cell lymphoma; GCB subtype: germinal center B cell-like subtype; non-GCB subtype: non-germinal center B cell-like subtype; ABC subtype: activated B cell-like subtype; BCL-2: B cell CLL / lymphoma 2; BCL-6: B cell CLL / lymphoma 6; DHL: double hit lymphoma; THL: triple hit lymphoma (DHL or THL refers to tumors with simultaneous rearrangement of MYC and BCL-2 and / or BCL-6); DEL: double expressor lymphoma. lymphoma), lymphoma with overexpression of MYC and BCL-2 proteins; HDAC inhibitors: histone deacetylase inhibitors.

[0008] MCD subtype: MYD88 L265P and CD79B mutations, mainly in the ABC subtype; BN2 subtype: BCL-6 translocation and NOTCH2 mutation, both in the ABC subtype and the GCB subtype; N1 type: NOTCH1 mutation, mainly seen in the ABC subtype; EZB-Myc positive type: with EZH2 mutation, BCL2 translocation, Myc positivity, mainly seen in the GCB subtype; EZB-Myc negative type: with EZH2 mutation, BCL2 translocation, Myc negativity, mainly seen in the GCB subtype; A53 type: TP53 mutation / deletion, more common in the ABC subtype; ST2 type: TET2 and SGK1 mutations, more common in the GCB subtype.

[0009] In a first aspect, the present invention provides the use of prilocastat mesylate and its analogs in the preparation of a medicament for preventing and / or treating diffuse large B-cell lymphoma.

[0010] The diffuse large B-cell lymphoma is selected from at least one of the GCB subtype, ABC subtype, non-GCB subtype or other unclassified subtypes.

[0011] The GCB subtype, ABC subtype, non-GCB subtype or other unclassified types all include subtype A and / or subtype B; the subtype A includes DLBCL subtypes in which one or more genes undergo at least one gene mutation, deletion, translocation, fusion, rearrangement and / or insertion; the subtype B includes at least one subtype of DLBCL NOS or DEL.

[0012] In subtype A, the gene that undergoes gene mutation, deletion, translocation, fusion, rearrangement and / or insertion is selected from at least one of MYD88 gene, CD79B gene, BCL-6 gene, NOTCH2 gene, NOTCH1 gene, EZH2 gene, BCL-2 gene, MYC gene, TP53 gene, TET2 gene, SGK1 gene, CREBBP gene, MEF2B gene, KMT2C gene, KMT2D gene, EP300 gene, IRF4 gene, HIST1H1E gene, ARID1A gene, DNA methylation gene TET2, PIM1 gene, KMT2D gene, CARD11 gene, BTG2 gene, ATM gene, RAG1 gene, CIITA gene, TNFRSF14 gene, NOTCH3 gene, FGFR3 gene, CCND3 gene, PCLO gene, HIST1H1C gene, PRDM1 gene, TMSB4X gene, LYN gene or SOCS1 gene.

[0013] In one embodiment of the present invention, the diffuse large B-cell lymphoma is selected from at least one of MCD subtype, BN2 subtype, N1 subtype, EZB-Myc positive / negative subtype, A53 subtype, ST2 subtype, DHL subtype and THL subtype.

[0014] In a preferred embodiment of the present invention, the diffuse large B-cell lymphoma is selected from at least one of DLBCL subtypes including DLBCL NOS, DHL, DEL lymphoma, TP53 mutation and / or deletion, TP53 mutation and / or deletion with dual expression, TP53 mutation and / or deletion with DHL, TP53 mutation and / or deletion with poor prognosis-associated gene mutation, or TP53 mutation and / or deletion with dual expression with poor prognosis-associated gene mutation; wherein the poor prognosis-associated genes include at least one of c-MYC, MYD88, BCL-2, CD79B, KMT2D, CARD11, BTG2, ATM, RAG1, CIITA, TNFRSF14, NOTCH3, TET2, FGFR3, CCND3, KMT2D, PIM1, PCLO, HIST1H1C, PRDM1, TMSB4X, LYN or SOCS1.

[0015] In a most preferred embodiment of the present invention, the diffuse large B-cell lymphoma is selected from TP53 mutation and BCL-2 rearrangement, ABC subtype (U2932 cell line); TP53 mutation and BCL-2 rearrangement, GCB subtype (SU-DHL-4, SU-DHL-6, Karpas 422 cell lines); c-MYC and BCL-2 rearrangement, GCB subtype (DOHH2 cell line); TP53 mutation, c-MYC and BCL-2 rearrangement, GCB subtype (OCI-LY18 cell line); TP53, MYD88 and CD79B mutation, ABC subtype (HBL-1 cell line); DLBCL NOS, GCB subtype (LY-24-0206PDX model); DEL, non-GCB subtype (LY-24-0179PDX model); DHL, GCB subtype (LY-24-0019PDX model); TP53 (R248Q), KMT2D, and PIM1 mutations, non-GCB subtype (LY-24-0004PDX model); TP53 (17P- and R175H-) deletions and mutations combined with KMT2D, CARD11, BTG2, ATM, RAG1, CIITA, and TNFR Non-GCB subtype with SF14, NOTCH3, BCL2, TET2, FGFR3 and CCND3 gene mutations (LY-24-0398 PDX model), TP53 mutation with dual expression, ABC subtype (xenograft mouse model established with U2932 cells) or TP53 (P300L) mutation with dual expression, combined with at least one of the GCB subtypes with CCND3, PCLO, HIST1H1C, TNFRSF14, PRDM1, TMSB4X, LYN, SOCS1 gene mutations (LY-24-0236 DLBCL PDX model).

[0016] In a second aspect, the present invention provides a combination drug of prilocastat mesylate and its analogs for preventing and treating diffuse large B-cell lymphoma. The combination drug is prilocastat mesylate and its analogs and other anti-tumor drugs administered separately or simultaneously.

[0017] The other anti-tumor drugs are selected from at least one of R-CHOP, Pola-R-CHP, R-CHP, ibrutinib, venetoclax, bendamustine, rituximab (RTX), tafasitamab, vepotuzumab (Pola), lenalidomide, and epcoritamab. Preferably, the other anti-tumor drugs are selected from at least one of RTX, R-CHOP, venetoclax, and R-CHP.

[0018] In one embodiment of the present invention, the combination drug comprises prilocastat mesylate and its analogs and rituximab as the main drugs. Preferably, the mass ratio of prilocastat mesylate and its analogs to rituximab is (1-2):(1-2). Most preferably, the mass ratio is 1:2.

[0019] In one embodiment of the present invention, the combination drug comprises prilocastat mesylate and its analogs and R-CHOP as the main drugs; wherein the R-CHOP is composed of rituximab, cyclophosphamide, daunorubicin, vincristine, and prednisolone in a mass ratio of 10:20:1.25:0.2:0.15. Preferably, the mass ratio of prilocastat mesylate and its analogs to R-CHOP is (1-2):(6-8). Most preferably, the mass ratio is 1:6.32.

[0020] In one embodiment of the present invention, the combination drug comprises Prilostat mesylate and its analogs, rituximab, and venetoclax as the main drugs, respectively. Preferably, the mass ratio of Prilostat mesylate and its analogs to rituximab and venetoclax is (1-2):(1-2):(6-10). Most preferably, the mass ratio of the main drugs is 1:2:8.

[0021] In one embodiment of the present invention, the combination drug comprises prilocastat mesylate and its analogs and R-CHP as the main drugs; wherein the R-CHP is composed of rituximab, cyclophosphamide, doxorubicin, and prednisolone in a mass ratio of 10:20:1.25:0.15. Preferably, the mass ratio of prilocastat mesylate and its analogs to R-CHP is (1-2):(6-8). Most preferably, the mass ratio is 1:6.28.

[0022] In a third aspect, the present invention provides use of the above-mentioned combination drug in preventing and / or treating diffuse large B-cell lymphoma.

[0023] The diffuse large B-cell lymphoma is selected from at least one of the GCB subtype, ABC subtype, non-GCB subtype or other unclassified subtypes.

[0024] The GCB subtype, ABC subtype, non-GCB subtype or other unclassified types all include subtype A and / or subtype B; the subtype A includes DLBCL subtypes in which one or more genes undergo at least one gene mutation, deletion, translocation, fusion, rearrangement and / or insertion; the subtype B includes at least one subtype of DLBCL NOS or DEL.

[0025] In subtype A, the gene that undergoes gene mutation, deletion, translocation, fusion, rearrangement and / or insertion is selected from at least one of MYD88 gene, CD79B gene, BCL-6 gene, NOTCH2 gene, NOTCH1 gene, EZH2 gene, BCL-2 gene, MYC gene, TP53 gene, TET2 gene, SGK1 gene, CREBBP gene, MEF2B gene, KMT2C gene, KMT2D gene, EP300 gene, IRF4 gene, HIST1H1E gene, ARID1A gene, DNA methylation gene TET2, PIM1 gene, KMT2D gene, CARD11 gene, BTG2 gene, ATM gene, RAG1 gene, CIITA gene, TNFRSF14 gene, NOTCH3 gene, FGFR3 gene, CCND3 gene, PCLO gene, HIST1H1C gene, PRDM1 gene, TMSB4X gene, LYN gene or SOCS1 gene.

[0026] In one embodiment of the present invention, in the use of the above-mentioned combination drug for the prevention and / or treatment of diffuse large B-cell lymphoma, the diffuse large B-cell lymphoma is selected from at least one of the MCD subtype, BN2 subtype, N1 type, EZB-Myc positive and negative type, A53 type, ST2 type, DHL type or THL type.

[0027] In a preferred embodiment of the present invention, in the use of the above-mentioned combination drug in the prevention and / or treatment of diffuse large B-cell lymphoma, the diffuse large B-cell lymphoma is selected from DLBCL At least one of the DLBCL subtypes of NOS, DHL, DEL lymphoma, TP53 mutation and / or deletion, TP53 mutation and / or deletion with dual expression, TP53 mutation and / or deletion with DHL, TP53 mutation and / or deletion with poor prognosis-related gene mutation, or TP53 mutation and / or deletion with dual expression with poor prognosis-related gene mutation; wherein the poor prognosis-related genes include at least one of c-MYC, MYD88, BCL-2, CD79B, KMT2D, CARD11, BTG2, ATM, RAG1, CIITA, TNFRSF14, NOTCH3, TET2, FGFR3, CCND3, KMT2D, PIM1, PCLO, HIST1H1C, PRDM1, TMSB4X, LYN or SOCS1.

[0028] In a most preferred embodiment of the present invention, in the use of the above-mentioned combination drug in the prevention and / or treatment of diffuse large B-cell lymphoma, the diffuse large B-cell lymphoma is selected from TP53 mutation and BCL-2 rearrangement, ABC subtype (U2932); TP53 mutation and BCL-2 rearrangement, GCB subtype (SU-DHL-4, SU-DHL-6, Karpas422); c-MYC and BCL-2 rearrangement, GCB subtype (DOHH2); TP53 mutation, c-MYC and BCL-2 rearrangement, GCB subtype (OCI-LY18); TP53, MYD88 and CD79B mutation, ABC subtype (HBL-1); DLBCL NOS, GCB subtype (LY-24-0206PDX model); DEL, non-GCB subtype (LY-24-0179PDX model); DHL, GCB subtype (LY-24-0019PDX model); TP53 (R248Q), KMT2D, and PIM1 mutations, non-GCB subtype (LY-24-0004PDX model); TP53 (17P- and R175H-) deletions and mutations combined with KMT2D, CARD11, BTG2, ATM, RAG1, CIITA, and TNFR Non-GCB subtype with SF14, NOTCH3, BCL2, TET2, FGFR3 and CCND3 gene mutations (LY-24-0398 PDX model), TP53 mutation with dual expression, ABC subtype (xenograft mouse model established with U2932 cells) or TP53 (P300L) mutation with dual expression, combined with at least one of the GCB subtypes with CCND3, PCLO, HIST1H1C, TNFRSF14, PRDM1, TMSB4X, LYN, SOCS1 gene mutations (LY-24-0236 DLBCL PDX model).

[0029] The aforementioned prilocastat mesylate and its analogs, or the aforementioned combination drug, is a preparation prepared with prilocastat mesylate and its analogs, or the combination drug with an anti-tumor drug as the active ingredient, and pharmaceutically acceptable excipients or auxiliary ingredients added thereto; preferably, the preparation is an injectable preparation. More preferably, the injection route is at least one of intravenous, intraperitoneal, intramuscular, subcutaneous, intradermal, or intramyocardial injection.

[0030] Wherein, the above-mentioned prilocastat mesylate and its analogs are selected from compounds having the following structures:

[0031] Preferably, the prilocastat mesylate and its analogs are selected from compounds having the following structures.

[0032] More preferably, the prilocastat mesylate and its analogs are prilocastat mesylate for injection.

[0033] More preferably, the clinical dose of Prilostat mesylate for injection is 5 mg / m 2 ~18 mg / m 2 .

[0034] Most preferably, the clinical dose of Prilostat mesylate for injection is 8.4 mg / m 2 ~11.2 mg / m 2 .

[0035] Beneficial Effects: Experiments in this invention demonstrate that PM, a novel, highly selective HDACI / IIb inhibitor, exerts anti-tumor efficacy by inhibiting the expression of key genes and proteins associated with DLBCL survival and activating in vivo anti-tumor immune responses. Its efficacy in treating high-grade DLBCL (DEL and DHL) PDX models with poor prognosis, PDX models with TP53 deletion and mutation combined with multiple prognostic gene mutations, and CDX and PDX models with TP53 mutation and dual expression all demonstrates excellent in vivo anti-tumor efficacy, outperforming various DLBCL clinical treatment options, including R-CHOP and selinexor.

[0036] The present invention also found that the dual or triple combination of PM with other anti-tumor drugs such as rituximab (RTX), R-CHOP, venetoclax, and R-CHP can also effectively prevent and treat DLBCL, especially for the treatment of high-grade DLBCL (DEL and DHL) PDX models with poor prognosis, PDX models with TP53 deletion and mutation combined with multiple poor prognosis gene mutations, and CDX and PDX models with TP53 mutation and dual expression, all of which have excellent in vivo anti-tumor efficacy.

[0037] These results indicate that PM has important clinical significance and academic value for DLBCL and its various subtypes. PM treatment is still relatively effective for refractory DLBCL subtypes with DHL, DEL, and TP53 mutations and / or deletions, while PM treatment is even more effective for general DLBCL subtypes. This expands the molecular mechanism network of class I and IIb HDAC inhibitors for the treatment of DLBCL, and lays a preclinical pharmacodynamic foundation for the Phase II clinical trial of PM for the treatment of R / R DLBCL.

[0038] Currently, PM has completed the Phase IIa clinical trial of PM for the treatment of R / R DLBCL. Existing data show that in human clinical trials, the efficacy of PM at 8.4 mg / m 2 and 11.2 mg / m 2Two dose groups were randomly divided into two groups, with 15 patients in each group, for a total of 30 patients. One treatment cycle was 21 days, and the drug was administered once on D1, 4, 8, and 11 in each cycle for 6 cycles. After 6 cycles, the researchers evaluated the benefit of the subjects and continued to receive the trial drug treatment. The frequency of continuous dosing was adjusted based on the results of the 6-cycle efficacy evaluation. As of July 28, 2024, 8.4 mg / m 2 15 patients were enrolled in the dose group, 15 of whom were evaluable, and 10 achieved clinical remission, with an ORR of 66.7% (10 / 15), including 1 complete remission (CR) and 9 partial remissions (PR). 2 Fifteen patients were enrolled in the dose group, 14 of whom were evaluable, and 10 achieved clinical remission, with an ORR of 71.4% (10 / 14), including 5 CRs and 5 PRs. Both dose groups showed an excellent trend in therapeutic efficacy, and no significant difference in safety was observed between the two dose groups. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 shows the expression analysis of HDAC1-11 isoforms in newly diagnosed DLBCL in Example 1; (A) HDAC class I; (B) HDAC class IIa; (C) HDAC class IIb; (D) HDAC class IV. *** indicates P < 0.001; ** indicates P < 0.01; vs. Normal, t-test.

[0040] Figure 2 shows the epigenetic profile and common DLBCL gene mutations of 1,295 newly diagnosed DLBCL patients from five research centers analyzed using the cBioportal database in Example 2; (A) Mutation ratios of genes related to epigenetic acetyltransferases, histone methyltransferases, chromatin remodeling, and DNA methylation; (B) Mutation ratios of TP53, MYD88, and PIM1, genes commonly mutated in DLBCL patients.

[0041] Figure 3 shows the IC50 values ​​of the antiproliferative activity of PM in Example 3.1 in different subtypes of human DLBCL cell lines after 72 hours of treatment; ABC: activated B cell subtype; GCB: germinal center B cell subtype; DHL: double hit lymphoma; realr: rearranged; wt: wild type.

[0042] FIG4 is a graph showing the anti-proliferative activities of PM, chidamide and LBH589 against seven human DLBCL cell lines in Example 3.1.

[0043] FIG5 is a graph showing the effect of PM exposure for 24 hours on the cell cycle of human DLBCL cell lines SU-DHL-4, DOHH2, and U2932 in Example 3.2.

[0044] Figure 6 shows the effects of PM and chidamide on SU-DHL-4 cell apoptosis in Example 3.3; (A) and (B) SU-DHL-4 cells were treated with different concentrations of PM and chidamide for 48 hours, and the proportion of cell apoptosis was detected by flow cytometry; (C) SU-DHL-4 cells were treated with different concentrations of PM and chidamide for 24 hours, and the expression changes of cell apoptosis-related proteins were detected by western blot.

[0045] Figure 7 shows the effects of PM and chidamide on U2932 cell apoptosis in Example 3.3; (A) and (B) flow cytometry analysis of the proportion of cell apoptosis after treating U2932 cells with different concentrations of PM and chidamide for 48 hours; (C) western blot analysis of the expression changes of apoptosis-related proteins after treating U2932 cells with different concentrations of PM and chidamide for 24 hours.

[0046] Figure 8 shows the effects of PM and chidamide on DOHH2 cell apoptosis in Example 3.3; (A) and (B) DOHH2 cells were treated with different concentrations of PM and chidamide for 48 hours, and the proportion of cell apoptosis was detected by flow cytometry; (C) DOHH2 cells were treated with different concentrations of PM and chidamide for 24 hours, and the expression changes of cell apoptosis-related proteins were detected by western blot.

[0047] Figure 9 shows the in vivo antitumor activity of PM in the dual-expressing DLBCL PDX (non-GCB) mouse model in Example 4.2; (A) tumor volume of mice during treatment; (B) weight changes of mice during treatment.

[0048] Figure 10 shows the in vivo anti-tumor activity of the combination of PM and R-CHOP in the dual-expressing DLBCL PDX (non-GCB) mouse model in Example 4.2, which is significantly superior to that of the combination of chidamide and R-CHOP; (A) Tumor volume of mice during treatment; (B) Weight changes of mice during treatment.

[0049] Figure 11 shows the in vivo antitumor activity of PM in the double-hit DLBCL PDX (GCB) mouse model treated with PM in Example 4.3; (A) tumor volume of mice during treatment; (B) weight changes of mice during treatment.

[0050] Figure 12 shows that the efficacy of PM in treating the double-hit DLBCL PDX (GCB) mouse model in Example 4.3 is significantly better than that of Selinexor: (A) tumor volume of mice during treatment; (B) weight changes of mice during treatment.

[0051] Figure 13 shows the effects of PM and other drug treatments in Example 4.4 on the organs of LY-24-0179 dual-expressing DLBCL PDX model mice (200X); black arrows indicate grade 1 degeneration of the heart, grade 3 degeneration of the renal tubules, and grade 3 intestinal epithelial cell shedding.

[0052] Figure 14 shows the effect of PM treatment on the MYC gene set in the tumor tissue of the dual-expression and dual-base DLBCL PDX models analyzed by Bulk RNA-seq and GSEA in Example 5.1; (A) and (B) LY-24-0179 (DEL, non-GCB) PDX model mice were given a single treatment of Vehicle and PM 5 mg / kg for 24 hours, and the MYC_TARGET_V1 and MYC_TARGET_V2 gene sets in the tumor tissue were significantly downregulated; (C) and (D) LY-24-0019 (DHL, GCB) PDX model mice were given a single treatment of Vehicle and PM 5 mg / kg for 24 hours, and the MYC_TARGET_V1 and MYC_TARGET_V2 gene sets in the tumor tissue were significantly downregulated.

[0053] Figure 15 shows that PM treatment in Example 5.1 significantly downregulated the expression of the poor prognosis protein c-MYC in tumor tissues of DLBCL cell lines and DLBCL PDX model mice; (A) SU-DHL-4, U2932 and DOHH2 diffuse large B cell lines were treated with different concentrations of PM for 24 hours, and the changes in c-MYC and MCL-1 protein expression were detected by western blot; (B) LY-24-0206 (DLBCL NOS, GCB) PDX model mice were continuously dosed for 16 days, and the changes in c-MYC protein expression were detected; LY-24-0179 and LY-24-0019 are DEL and DHL DLBCL PDX models, respectively. Long-term administration of PM resulted in complete disappearance of tumor tissue in most mice, so the model was re-established, with 3 mice in each group, including the Control group, PM 5 mg / kg group, PM 5 mg / kg+RTX group, and Chi 12.5 mg / kg group. After 24 hours of a single dose, tumor tissue was collected to detect changes in c-MYC protein levels.

[0054] Figure 16 shows the effect of a single 24-hour treatment of Vehicle and PM 5 mg / kg on the key gene sets for survival of tumor cells in the dual-expressing DLBCL PDX (LY-24-0179) model as analyzed by bulk RNA-seq and GSVA in Example 5.2; the left side shows a heat map, and the right side shows a bar chart, with red indicating upregulated gene sets and blue indicating downregulated gene sets.

[0055] Figure 17 shows the effect of a single 24-hour treatment with 5 mg / kg of Vehicle and PM on the key gene sets for survival of tumor cells in the double-hit DLBCL PDX (LY-24-0019) model, as analyzed by bulk RNA-seq and GSVA in Example 5.2; the left side shows a heat map, and the right side shows a bar chart, with red indicating upregulated gene sets and blue indicating downregulated gene sets.

[0056] Figure 18 is a GSEA diagram of Example 5.2 showing that PM 5 mg / kg treatment significantly enriched and downregulated the E2F, G2M checkpoint, unfolded protein and DNA repair and oxidative phosphorylation, and MTORC1 signaling pathway gene sets in tumor tissues of dual-expression DLBCL PDX model mice (LY-24-0179).

[0057] Figure 19 is a GSEA diagram showing that PM 5 mg / kg treatment significantly enriched and downregulated the E2F, G2M checkpoint, unfolded protein and DNA repair and oxidative phosphorylation, and MTORC1 signaling pathway gene sets in tumor tissues of double-hit DLBCL PDX model mice (LY-24-0019) in Example 5.2.

[0058] Figure 20 is a graph showing the double-expression DLBCL PDX (LY-24-0179) model treated with a single dose of PM 5 mg / kg in Example 5.3. GSEA shows that the gene sets "HALLMARK_TNFA_SIGNALING_VIA_NFKB", "HALLMARK_INFLAMMATORY_RESPONSE", "HALLMARK_IL2_STAT5_SIGNALIN", "HALLMARK_HYPOXIA", "HALLMARK_COMPLEMENT" and "HALLMARK_INTERFERON_GAMMA_RESPONSE" related to inflammation and interferon response in tumor tissues are significantly upregulated.

[0059] Figure 21 is a graph of the double-hit DLBCL PDX (LY-24-0019) model treated with a single dose of PM 5 mg / kg in Example 5.3. GSEA shows that the gene sets "HALLMARK_TNFA_SIGNALING_VIA_NFKB", "HALLMARK_INFLAMMATORY_RESPONSE", "HALLMARK_IL2_STAT5_SIGNALIN", "HALLMARK_HYPOXIA", "HALLMARK_COMPLEMENT" and "HALLMARK_INTERFERON_GAMMA_RESPONSE" related to inflammation and interferon response in tumor tissues are significantly upregulated.

[0060] Figure 22 is a graph showing that GSEA showed significant enrichment and upregulation of gene sets such as T cell activation, proliferation and differentiation after a single treatment of the dual-expressing DLBCL PDX (LY-24-0179) model with PM 5 mg / kg in Example 5.3.

[0061] Figure 23 is a graph showing significant enrichment and upregulation of gene sets such as T cell activation, proliferation, and differentiation after a single dose of PM 5 mg / kg in the double-hit DLBCL PDX (LY-24-0019) model in Example 5.3, as shown by GSEA.

[0062] Figure 24 shows the anti-tumor activity of PM on the A20 mouse model and the effect of T cell subtypes in tumor tissues in Example 5.4; (A) tumor volume of mice during treatment; (B) tumor weight of mice in each group after mice were sacrificed on the 14th day; (C) and (D) flow cytometry analysis of the ratio of CD4+ cells and CD8+ cells in CD3+ lymphocytes in tumor tissues of each treatment group; (E) and (F) flow cytometry analysis of the ratio of CD44+CD62L- cells in CD4+ and CD8+ lymphocytes in tumor tissues of each treatment group; compared with the control group, NS indicates P>0.05; * indicates P<0.05, ** indicates P<0.01; **** indicates P<0.0001, t-test.

[0063] Figure 25 is a graph showing that bulk RNA-Seq in Example 5.5 shows that a single treatment with 5 mg / kg of PM significantly upregulated the expression of proteins encoded by MHC class I and MHC class II molecules in tumor tissues of DEL and DHL DLBCL PDX model mice.

[0064] Figure 26 is a GSEA diagram showing that a single dose of PM 5 mg / kg significantly enriched and upregulated the MHC class I protein binding, MHC class II protein binding, and MHC II protein complex binding gene sets in tumor tissues of DEL DLBCL PDX (LY-24-0179) model mice in Example 5.5.

[0065] FIG27 is a GSEA diagram showing that a single dose of PM 5 mg / kg significantly enriched and upregulated the MHC class I protein binding, MHC class II protein binding, and MHC II protein complex binding gene sets in tumor tissues of DHL DLBCL PDX (LY-24-0019) model mice in Example 5.5.

[0066] Figure 28 shows the concentration-dependent upregulation of MHC I and MHC II expression on the surface of the DLBCL cell line U2932 cells in Example 5.5 by PM: (A) and (B) Flow cytometric analysis of MHC-I expression on the surface of U2932 cells treated with different concentrations of PM and chidamide for 24 hours; (C) and (D) Flow cytometric analysis of MHC-II expression on the surface of DOHH2 cells treated with different concentrations of PM and chidamide for 24 hours. Compared with the control group, NS indicates P > 0.05; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001, t-test.

[0067] Figure 29 is a graph showing the acetylation levels of histones and P53 mutant proteins in TP53 mutant cell lines U2932 and SU-DHL-4 detected by western blot after treatment with different concentrations of PM for 24 hours in Example 6.1.

[0068] Figure 30 shows the antitumor activity of PM in the TP53 (R248Q) mutant DLBCL PDX mouse model described in Example 6.2; (A) Tumor volume during treatment; (B) Body weight change during treatment; (C) Tumor weight after 27 days of treatment; (D) Tumor size in each treatment group. "-" indicates complete tumor regression. * indicates P < 0.05; ** indicates P < 0.01 compared with the control group, t-test.

[0069] Figure 31 shows the antitumor activity of PM in the TP53 (17P- and R175H)-deficient and mutant DLBCL PDX mouse models described in Example 6.3; (A) Tumor volume during treatment; (B) Body weight change during treatment; (C) Tumor weight after 20 days of treatment; (D) Tumor size in each treatment group. "-" indicates complete tumor regression. NS indicates P > 0.05; *** indicates P < 0.001; **** indicates P < 0.0001; ## indicates P < 0.01; #### indicates P < 0.0001 compared to the PM 5 mg / kg group, t-test.

[0070] Figure 32 shows the antitumor activity of PM in the U2932 mouse model of TP53-mutant DLBCL cells described in Example 6.4; (A) Tumor volume during treatment; (B) Body weight change during treatment; (C) Tumor weight after 18 days of treatment; (D) Tumor size in each treatment group. "-" indicates complete tumor regression. NS indicates P > 0.05 compared to the control group; *** indicates P < 0.001; **** indicates P < 0.0001; #### indicates P < 0.0001 compared to the PM 5 mg / kg group, t-test.

[0071] Figure 33 shows the antitumor activity of PM in the TP53 (P300L) mutant and dual-expressing DLBCL PDX mouse model described in Example 6.5; (A) Tumor volume during treatment; (B) Body weight change during treatment; (C) Tumor weight after 14 days of treatment; (D) Tumor size in each treatment group. "-" indicates complete tumor regression. NS indicates P > 0.05; *** indicates P < 0.001 compared to the control group.

[0072] Figure 34 shows the efficacy of PM in the Phase IIa clinical trial of R / R DLBCL patients in Example 6.6; (A) Lane diagram of the Phase IIa clinical trial of PM in 29 patients with R / R DLBCL; (B) Statistical diagram of the efficacy of PM in the Phase IIa clinical trial of 29 patients with R / R DLBCL (complete remission (CR), partial remission (PR), stable disease (SD), progressive disease (PD)). DETAILED DESCRIPTION

[0073] Clinically, patients with high-grade B-cell lymphoma (HBCL) with MYC abnormalities and DLBCL with TP53 abnormalities are resistant to conventional chemotherapy and often have a poor prognosis. Patients with TP53 mutations and dual-expression DLBCL with high MYC and BCL-2 expression have extremely poor treatment responses.

[0074] PM for injection is a highly selective and active HDACI / IIb inhibitor with superior inhibitory activity against HDACI / IIb enzymes compared to all currently marketed HDAC inhibitors. Based on the current unmet clinical needs of DLBCL treatment, this study investigates the preclinical pharmacodynamics and mechanism of action of PM, a novel Class I / IIb HDAC inhibitor, in the treatment of DLBCL. The main implementation details are as follows:

[0075] (1) Analyze the expression differences of HDAC 1-11 in normal human peripheral blood leukocytes and newly diagnosed DLBCL tumor tissues through public databases, and conduct epigenetic and TP53 mutation analysis in DLBCL patients to provide a basis for the treatment of DLBCL with HDACI / IIb inhibitors;

[0076] (2) In vitro anti-tumor activity studies of PM in DLBCL cell lines, including DEL and DHL high-grade lymphomas, and cells harboring mutations in various DLBCL genes with poor prognosis, such as TP53, including anti-proliferative activity, apoptosis induction, and cell cycle arrest;

[0077] (3) To investigate the in vivo antitumor activity of PM alone at different doses and in combination with rituximab (RTX) in DLBCL PDX models, including three PDX models: DLBCL NOS (GCB, LY-24-0206), DEL (non-GCB, LY-24-0179), and DHL (GCB, LY-24-0019). The first-line treatment regimen R-CHOP (rituximab, cyclophosphamide, daunorubicin, vincristine, and prednisolone) and the new drug XPO1 inhibitor selinexor were used as positive controls to compare the efficacy and toxicity. In the DEL (non-GCB, LY-24-0179) PDX model, the efficacy and toxicity of PM + R-CHOP and chidamide + R-CHOP were further explored.

[0078] (4) Exploration of the mechanism of PM treatment for DLBCL, including: ① RNA-seq of tumor tissues of DEL (non-GCB, LY-24-0179) and DHL (GCB, LY-24-0019) model mice treated with vehicle and PM, and bioinformatics analysis of the effects of PM treatment on key genes and signaling pathways for DLBCL cell survival; ② Investigating the effects of PM treatment on the expression of oncoproteins such as c-MYC, a key biomarker for poor prognosis in DLBCL, through PM-treated cell lines and model mouse tumor tissues; ③ Investigating the effects of PM treatment on in vivo anti-tumor immunity through transcriptome sequencing and the DLBCL A20 mouse model with an immune system; ④ Investigating the effects of PM treatment on the expression of MHC I and MHC II in tumor cells through transcriptome sequencing combined with PM-treated DLBCL cell lines;

[0079] (5) Further explore the effect of PM treatment on the expression of TP53 mutant protein and Ac-P53 protein using PM-treated DLBCL cell lines and PDX model tumor tissues;

[0080] (6) In vivo anti-tumor activity study of PM in TP53-deficient and mutated DLBCL and TP53-mutated DLBCL PDX models, including: ① DLBCL with TP53 (R248Q) mutation combined with KMT2D and PIM1 mutations ② The PDX (LY-24-0004) model, in which the tumor tissue pathology showed BCL-2 (+), was used as a positive control to investigate the efficacy and toxicity differences of PM, a new first-line treatment for DLBCL, with Pola+R-CHP and Venetoclax+R-CHOP. ② The PDX (LY-24-0398) model, in which TP53 (17P- and R175H) deletions and mutations coexist and multiple DLBCL gene mutations with poor prognosis were combined. Pola-R-CHP and BEBT-908 were used as positive controls to investigate the efficacy and toxicity differences of PM alone and PM combined with R-CHP. ③ R-CHOP was used as a positive control to investigate the efficacy of PM alone and PM combined with RTX in the treatment of DLBCL with TP53 mutation and dual expression. The differences in efficacy and toxicity of the U2932 mouse model; ④ The DLBCL PDX (LY-24-0236) model with TP53 (P300L) mutation and dual expression, and combined with multiple DLBCL gene mutations with poor prognosis, using Pola + R-CHP and cedabendine as positive controls, to investigate the differences in efficacy and toxicity of PM alone and PM combined with R-CHP.

[0081] In summary, this study targets high-grade and poor-prognosis DLBCL subtypes with unmet clinical needs, including: ① DEL and DHL high-grade DLBCL; ② DLBCL with TP53 deletion, mutation, and multiple common DLBCL mutations; and ③ TP53 mutation with dual-expression DLBCL, exploring the efficacy and mechanism of action of PM. Furthermore, during the study, in vivo efficacy was explored using highly representative cell lines, CDX mouse models, and PDX mouse models (a total of 6 mouse models for different subtypes), and the efficacy and toxicity were compared with a variety of existing clinical drugs. Mechanistically, PM-treated cell lines, PDX mouse models, and DLBCL mouse models with an immune system were combined with bulk RNA-seq and various pharmacological approaches to investigate the transcription and protein expression of genes implicated in poor prognosis, as well as anti-tumor immunity. This study has important academic value and clinical significance, expanding the molecular mechanism network of class I and IIb HDAC inhibitors in the treatment of DLBCL, and laying an important clinical foundation for the Phase II clinical trial of PM in the treatment of R / R DLBCL. Currently, PM has completed the Phase IIa clinical trial of PM for the treatment of R / R DLBCL. Existing data show that in human clinical trials, the efficacy of PM at 8.4 mg / m 2 and 11.2 mg / m 2Two dose groups were randomly divided into two groups, with 15 patients in each group, for a total of 30 patients. One treatment cycle was 21 days, and the drug was administered once on D1, 4, 8, and 11 in each cycle for 6 cycles. After 6 cycles, the researchers evaluated the benefit of the subjects and continued to receive the trial drug treatment. The frequency of continuous dosing was adjusted based on the results of the 6-cycle efficacy evaluation. As of July 28, 2024, 8.4 mg / m 2 15 patients were enrolled in the dose group, 15 of whom were evaluable, and 10 achieved clinical remission, with an ORR of 66.7% (10 / 15), including 1 CR and 9 PR. 2 Fifteen patients were enrolled in the dose group, 14 of whom were evaluable, and 10 achieved clinical remission, with an ORR of 71.4% (10 / 14), including 5 CRs and 5 PRs. Both dose groups showed an excellent trend in therapeutic efficacy, and no significant difference in safety was observed between the two dose groups.

[0082] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0083] 1. The reagents and materials used in the examples are shown in Table 1:

[0084] Table 1 Reagent and material information

[0085] Prilostat mesylate structural formula, molecular formula: C 23 H 26 N 10 O3·CH4O3S, molecular weight: 586.62.

[0086] 2. Experimental cell lines and cultures used in the examples:

[0087] 1) The human DLBCL cell lines U2932, SU-DHL-4, SU-DHL-6, DOHH2, Karpas422, and OCI-LY18 used in this experiment were purchased from the American Type Culture Collection (ATCC); the HBL-1 cell line was from Peking University Cancer Hospital; and the murine B-cell lymphoma A20 cell line was purchased from ATCC. All cell lines were authenticated before use and maintained in liquid nitrogen tank A in the Quality Control Room of the Molecular Biology Laboratory, Natural Products Building, State Key Laboratory of Biotherapy, Sichuan University.

[0088] 2) SU-DHL-4, SU-DHL-6, DOHH2, Karpas 422, and OCI-LY18 cell lines were cultured in RPMI-1640 complete medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / L streptomycin. HBL-1 cell line was cultured in RPMI-1640 complete medium supplemented with 20% fetal bovine serum, 1% non-essential amino acids (NEAA), 100 U / mL penicillin, and 100 mg / L streptomycin. A20 cell line was cultured in RPMI-1640 complete medium supplemented with 10% fetal bovine serum, 0.05 mM β-mercaptoethanol, 100 U / mL penicillin, and 100 mg / L streptomycin. These cell lines were cultured in a 37°C, 5% CO2 incubator. Cells were grown in suspension and passaged every 2-3 days depending on cell growth.

[0089] 3. The information of experimental animals used in the examples is as follows:

[0090] The animal experiment protocol for this study adhered to the 3R principles and was approved by the Experimental Animal Ethics Committee of West China Hospital, Sichuan University. For all DLBCL tumor-bearing mouse models, the tumor diameter (d) did not exceed 20 mm.

[0091] Cryopreserved tissues for the LY-24-0206, LY-24-0179, LY-24-0019, LY-24-0398, and LY-24-0004 PDX models were obtained from Shanghai Ruijin Hospital. All PDX models were maintained in liquid nitrogen tank B in the Quality Control Laboratory of the Molecular Biology Laboratory, Natural Products Building, State Key Laboratory of Biotherapy, Sichuan University.

[0092] LY-24-0206DLBCL PDX model, LY-24-0179DLBCL PDX model, and LY-24-0019DLBCL PDX model: SPF-grade NOD-SCID female mice, 5-6 weeks old, weighing 18-20 g, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (SCXK (Beijing) 2021-0006). Mice were housed in the SPF animal room of the State Key Laboratory of Biotherapy, Sichuan University.

[0093] LY-24-0398DLBCL PDX model, LY-24-0004DLBCL PDX model, and LY-24-0236DLBCL PDX model: SPF-grade NTG female mice, 5-6 weeks old, weighing 18-20 g, were purchased from Beijing Sibeifu Biotechnology Co., Ltd. (SCXK (Beijing) 2019-0010). Mice were housed in the SPF animal room of the Animal Experimental Center, West China Hospital, Sichuan University.

[0094] U2932 mouse subcutaneous tumor model: NOD-SCID female mice, 5-6 weeks old, 18-20 g, were purchased from Beijing Huafukang Biotechnology Co., Ltd. (SCXK (Beijing) 2019-0008) and housed in the SPF animal room of the State Key Laboratory of Biotherapy, Sichuan University.

[0095] A20 mouse subcutaneous tumor and systemic lymphoma model: Female Balb / c mice, 6 weeks old, weighing approximately 20 g, were purchased from Beijing Sibeifu Biotechnology Co., Ltd. (SCXK (Beijing) 2019-0010). Mice were housed at the Animal Experimental Center of West China Hospital, Sichuan University.

[0096] All the animals were adaptively raised for 1 week before the experiment.

[0097] 4. Experimental methods used in the examples:

[0098] 4.1 Source and Processing of DLBCL Patient Data

[0099] The data from newly diagnosed DLBCL patients selected for this example were obtained from the TCGA (The Cancer Genoma Atlas) database, and the data from normal human peripheral blood leukocytes were obtained from the GTEx (Genotype-Tissue Expression) database. The TCGA database contains RNA-seq results from 47 newly diagnosed DLBCL patients, while the GTEx database contains RNA-seq data from 444 normal human peripheral blood leukocytes. These RNA-seq data were downloaded from UCSC Xena (https: / / xena.ucsc.edu / ) and uniformly processed using the Toil pipeline. Based on the extracted RNA-seq data, the expression differences of HDAC class I (HDAC 1, HDAC 2, HDAC 3, and HDAC 8), HDAC class IIa (HDAC 4, HDAC 5, HDAC 7, HDAC 9), HDAC class IIb (HDAC 6, HDAC 10), and HDAC class IV (HDAC 11) were compared between normal human peripheral blood leukocytes and DLBCL patient tumor tissues.

[0100] 4.2 Detection of cell proliferation activity by CCK8 assay

[0101] Take human DLBCL cells in the logarithmic growth phase, centrifuge at 500g, add complete medium to resuspend the cells, and then count. Cells were seeded in 96-well plates, with approximately 15,000-30,000 cells per well. The volume of culture medium containing cells was 100 μL. At the same time, blank control wells and solvent control wells were set up. The side wells were filled with sterile saline and placed in a 37°C incubator for continued culture. The same culture medium was used to dilute the drug according to the drug concentration. The prepared drugs of different concentrations were added to the corresponding 96-well plates at 100 μL per well. 100 uL of the same culture medium was added to each well of the blank group, and the final volume per well was 200 μL. 200 μL of culture medium containing the same amount of DMSO as the highest drug concentration was added to the solvent control group. Three replicates were set up for each group and continued to be incubated in a 37°C, 5% CO2 cell culture incubator.

[0102] After 48h and 72h of cell culture, 20μL of CCK8 working solution was added to each well and cultured in a 37°C incubator for 1 to 4 hours before terminating the culture. The absorbance at a wavelength of 450nm was detected on a microplate reader. The cell viability was calculated using the formula: cell viability % = [(average OD value of the experimental group - average OD value of the solvent group) / (average OD value of the blank control group - average OD value of the solvent group)] × 100%. Finally, the cell viability of different drug concentrations was calculated, and then the IC was fitted using Graphpad Prism software. 50 Curve Calculation IC 50 value.

[0103] Calculate the inhibition rate of cell proliferation activity at different drug concentrations, and then use Graphpad Prism software to fit IC 50 value.

[0104] 4.3 Flow cytometry detection of cell cycle

[0105] The U2932, SU-DHL-4 and DOHH2 cells that were growing well were counted and seeded into 6-well plates, with approximately 1×10 cells per well. 6 cells and continue incubation. The drug was then prepared according to the drug concentration, and 1 mL was added to the corresponding wells of the 6-well plate to make the final concentration of PM 0.5 nM, 1 nM, and 5 nM, and the final concentration of cedabendine was 500 nM, 1500 nM, and 4500 nM. The 6-well plate was placed in a 37 ° C, 5% CO2 cell culture incubator and incubated for 24 hours. After 24 hours, the 6-well plate was removed, the cells were collected, the cells were washed once with pre-cooled PBS, centrifuged at 300g for 5 minutes, and the supernatant was discarded. The cell pellet was then placed on a vortex instrument and gently vortexed and 500 μL of pre-cooled 75% ice ethanol was slowly added to fix it, so that it was fully mixed and prevented from clumping. It was placed at 4 ° C overnight.

[0106] To prepare PI working solution: Weigh 1.0 mg of PI powder and dissolve it in PBS by vortexing. Add 100 μL of Triton-100 and mix thoroughly. Weigh 20.0 mg of RNase A powder and add it to the solution. Mix gently and dilute to 20 mL with PBS (final concentration: 50 ng / mL PI, 1 mg / mL RNase A, 0.5% Triton-100).

[0107] Centrifuge the fixed cells at 1500 rpm for 3 minutes, discard the supernatant, and wash the cells twice with PBS, removing as much of the supernatant as possible. Add 500 μL of PI working solution to each tube and mix thoroughly by pipetting carefully with a pipette. Stain for 10 minutes at room temperature in the dark. Filter through a 300-mesh filter and analyze the cell cycle using a flow cytometer.

[0108] 4.4 Annexin V / PI double staining to detect cell apoptosis

[0109] The cell status was observed under a microscope, and the U2932, SU-DHL-4, and DOHH2 cells in good condition were counted. 1×10 cells were seeded per well of a 6-well plate. 6 , and added different concentrations of PM and cedabendine to make the final concentrations of PM 1, 2.5, 5 and 10 nM, and the final concentrations of cedabendine 500, 100 and 2000 nM. The cells were incubated for another 48 hours. After the incubation, the experiment was performed according to the instructions of the Yisheng Cell Apoptosis Detection Kit. Briefly, after 48 hours, the cells were harvested and washed with PBS, and 100 μL of 1× Binding Buffer was added to each tube to resuspend the cells. Then, 5 μL of Annexin V-FITC and 10 μL of PI Staining solution were added to each sample, pipetted to mix, and incubated at room temperature in the dark for 15 minutes. Finally, an appropriate amount of Binding Buffer was added to each sample tube, placed on ice, and waited for detection on the machine. After the flow cytometer test, the results were analyzed using Flowjo software.

[0110] 4.5 In vivo experiments with DLBCL patient-derived xenograft (PDX) models

[0111] 4.5.1 LY-24-0206PDX (DLBCL NOS, GCB) Mouse Model

[0112] The immunohistochemistry results of the patients corresponding to this model were: tumor cell CK (-) CD20 (+), CD79α (+), CD10 (foci +), CD5 (-), CyclinD1 (-), CD21 (-), Bc16 (small foci +), c-myc (30% +), MUM-1 (20%), CD30 (-), Ki67 (80%), P53 (+).

[0113] (1) Pre-treatment for large-scale inoculation: Take out the frozen tissue of the model from the corresponding liquid nitrogen tank and place it in a 37°C water bath to thaw as soon as possible. Then place it in a clean bench, wash twice with sterile PBS, centrifuge at 500g for 5 minutes, discard the supernatant, place the tumor tissue block in a 50mL BD tube, and place it on ice. Enter the animal room and inoculate the mouse under the right armpit. After inoculation, observe the tumor formation of the mouse. After the mouse has formed a tumor, the tumor tissue is about 1000mm 3 When the tumor tissue was about 1 hour old, it was taken out again. A part of it was sent for pathology, immunohistochemistry and FISH detection to verify the model, and the other part was used for passage and seed preservation.

[0114] (2) Subcutaneous inoculation of tumors in NOD-SCID mice: The results of tumor tissue pathology and immunohistochemistry in the model mice were basically consistent with those of the corresponding patients, confirming that the model was successfully constructed. 3 Around 10:00 p.m., large-scale vaccinations were performed. First, enter the animal room, routinely disinfect the clean bench and all equipment used. Tumor-bearing mice were sacrificed, and the tumor tissue was cut into mung bean-sized pieces. Then, the mice were inoculated under the right armpit using a vaccination needle. After vaccination, the mice were returned to their cages and their condition, skin abnormalities at the inoculation site, and wound healing were closely observed.

[0115] (3) Grouped administration: After the tumors were formed in mice, the tumor volume reached 200-300 mm 3 Random grouping,

[0116] The model was divided into 7 groups according to the principle of 6 mice per group, namely, Control group, PM 5 mg / kg group, PM 10 mg / kg group, PM 5 mg / kg + RTX 10 mg / kg group, R-CHOP group, PM 5 mg / kg + R-CHOP group, and Selinexor 10 mg / kg group. The specific conditions and dosing schedules are shown in Tables 2 and 3.

[0117] (4) Drug preparation

[0118] PM: Prilostat mesylate for injection, 20 mg / vial, add 5 mL of normal saline to make 4 mg / mL, store in a refrigerator at 4°C, use within one week;

[0119] RTX: rituximab injection, 10 mg / mL, diluted in normal saline;

[0120] Cyclophosphamide: powder, prepared with normal saline before use;

[0121] Doxorubicin injection: 2 mg / mL, prepared with normal saline before use;

[0122] Vincristine: powder, reconstituted with normal saline before use;

[0123] Prednisolone: ​​powder, reconstituted with normal saline before use;

[0124] Selinexor: powder, prepared with 1% DMSO + 20% PEG400 + 2.5% Tween-80 before use.

[0125] (5) Detection indicators: mainly observation of mouse status, mouse weight and detection of mouse tumors.

[0126] Observe the condition of the mice: During treatment, closely monitor the condition of the mice, including general performance and any abnormal performance. If any abnormality is found, detailed records should be kept.

[0127] Mouse body weight detection: Weigh the mice every 2-3 days.

[0128] Detection indicators of mouse tumors: Use a vernier caliper to measure the long diameter and short diameter of the mouse tumor three times a week, calculate the tumor volume, and make a record.

[0129] (6) Sacrifice and dissection of mice: At the end of the experiment, mice were anesthetized with 2.5% avertin solution and sacrificed by cervical dislocation. Tumor tissues were removed from the mice using dissection tools, weighed, and photographed. Three mice in each group were selected to obtain the heart, liver, spleen, lung, kidney, stomach, and intestine, fixed with paraformaldehyde, and sent for pathological examination.

[0130] (7) Data processing: The main indicators of this animal experiment include the following: tumor volume, relative tumor volume (RTV), relative tumor proliferation rate (Treatment / Control Index, T / C) and tumor inhibition rate (%). The specific calculation formula is:

[0131] Tumor volume (TV) (mm 3 ) = tumor long diameter × tumor short diameter 2 / 2;

[0132] Relative tumor volume (RTV) = V t / V0 (where V0 is the tumor volume on day 0, and Vt is the tumor volume of mice in this group on day t);

[0133] Relative tumor proliferation rate (T / C) = T RTV / C RTV ×100%(T RTV Relative tumor volume of treatment group, C RTV is the relative tumor volume of the Control group);

[0134] Tumor inhibition rate (%) = [average tumor weight of the control group (g) - average tumor weight of the drug-treated group (g)] / average tumor weight of the control group (g) × 100%;

[0135] The efficacy evaluation criteria were: T / C > 60% was considered ineffective; T / C ≤ 60% with P < 0.05 as statistically analyzed was considered effective. A tumor inhibition rate < 40% was considered ineffective, and a tumor inhibition rate ≥ 40% with P < 0.05 was considered effective. As long as either T / C or tumor inhibition rate met the effective criteria, the treatment was considered effective.

[0136] Table 2 Grouping and dosing regimen of LY-24-0206PDX model mice

[0137] Table 3 R-CHOP dosing regimen

[0138] 4.5.2 LY-24-0179PDX (DEL, non-GCB) mouse model

[0139] Pretreatment for large-scale inoculation and subcutaneous inoculation of NOD-SCID mice with tumors were the same as described in 4.5.1. Immunohistochemistry results for the corresponding patient in this model were: CD20+, Bcl-2 (approximately 90%), C-myc+ (approximately 40%), PAX-5+, CD3-, CD5-, CK-, scattered weakly positive BCl-6, CD10-, Mum-1+, Ki-67+ (approximately 80%), and clinically confirmed DEL. Pathology and immunohistochemistry of tumor tissue in the model mice were generally consistent with those in the corresponding patient, and FISH revealed no rearrangement of Myc and BCL-2, demonstrating the successful establishment of this model.

[0140] The animal experiment of this model was divided into two batches. The first batch: mice with tumor volume of 150mm 3 The mice were divided into 6 groups: Control group, PM 5mg / kg group, PM 10mg / kg group, PM 5mg / kg + RTX 10mg / kg group, R-CHOP group, and Selinexor 10mg / kg group. The specific dosing regimen and grouping are shown in Table 4. The second batch: Because of PM or Chidamide combined with R-CHOP treatment, when the tumor volume of mice reached 300-400mm 3 The drugs were divided into three groups: Control group, PM+R-CHOP group and Chi+R-CHOP group. The specific dosing schedule is shown in Table 5.

[0141] Drug preparation: 1) PM, RTX, cyclophosphamide, doxorubicin, vincristine, and prednisolone should be diluted or prepared with normal saline. The specific formula of Selinexor is shown in 4.5.1.

[0142] 2) Chidamide: Powder, prepared using 0.2% sodium carboxymethylcellulose (CMCNa) and 0.1% Tween-80 aqueous solution. The solvent preparation is as follows: first, weigh 0.5g of CMCNa and dissolve it in 250mL of UP water. Heat and stir continuously to dissolve. Finally, add 250μL of Tween-80. After packaging, store in a refrigerator at 4°C.

[0143] Otherwise, the test parameters, killing and dissecting mice, and data processing are the same as in 4.5.1.

[0144] Table 4 LY-24-0179PDX model mouse grouping and dosing regimen (first batch)

[0145] Table 5 Grouping and dosing regimen of LY-24-0179PDX model mice (second batch)

[0146] 4.5.3 LY-24-0019PDX (DHL, GCB) Mouse Model

[0147] The pre-treatment and subcutaneous inoculation of tumors in large-scale inoculations of NOD-SCID mice were the same as in 4.5.1. The model was successfully established through pathological and immunohistochemical verification, and FISH results from mouse tumor tissues indicated rearrangements of Myc and BCL-2, confirming that the model was DHL. This part of the experiment was also completed in two batches. The first batch: mice with tumors of 100-200 mm 3 The mice were divided into 5 groups: Control group, PM 5mg / kg group, PM 10mg / kg group, PM 5mg / kg+RTX 10mg / kg group, and R-CHOP group. The specific dosing regimen and grouping are shown in Table 6. The second batch: When the tumor volume of mice reached 100-200mm 3 The mice were divided into three groups: control group, PM 5 mg / kg group, and selinexor group. The specific dosing schedule is shown in Table 7. The drug preparation, test parameters, mouse sacrifice and dissection, and data processing were the same as in 4.5.1.

[0148] Table 6 LY-24-0019PDX model mouse grouping and dosing regimen (first batch)

[0149] Table 7 LY-24-0019PDX model mouse grouping and dosing regimen (second batch)

[0150] 4.5.4 LY-24-0398PDX (non-GCB, TP53 mutation) mouse model

[0151] The immunohistochemical results of the model mice corresponding to the patients were CD20(+), CD79α(+), CD10(+), Bcl-6(+), Bcl-2(-), CD21(dendritic+), CD23(+), CD3(-), CD5(-), Cyclin D1(-), Ki67(approximately >90%+), CD30(-), CD19(+), CD22(positive), Myc(50%+), ALK(-), AE1 / AE3(-); EBER in situ hybridization: (-). NSG results suggest: 1) Mutation points related to disease type were detected: KMT2D, CARD11, BTG2; 2) Mutation points related to disease prognosis were detected: TP53; 3) Mutation points related to treatment were detected: ATM, CARD11; 4) Other mutations that may be related to the disease: BTG2, RAG1, CIITA, TNFRSF14, NOTCH3, BCL2, TET2, FGFR3, CCND3. The TP53 mutation site in this patient was R175H.

[0152] The pre-inoculation treatment, test parameters, killing and dissecting mice, and data processing of large-scale inoculation are the same as those in 4.5.1. Since NOD-SCID and NTG mice were used for pre-inoculation of this model, and NTG mice have a higher tumor formation rate, NTG mice were selected for large-scale inoculation. The tumor volume of mice was 100-200mm 3 As the Pola + R-CHP regimen has been approved for first-line treatment of DLBCL, this study used Pola + R-CHP as a positive control. Six groups were divided into the control group, the PM 5 mg / kg group, the PM 10 mg / kg group, the Pola + R-CHP group, and the PM + R-CHP group, with six mice in each group. Specific grouping and dosing schedules are shown in Tables 8 and 9.

[0153] Drug preparation: The preparation of PM, RTX, cyclophosphamide, doxorubicin, and prednisolone is the same as in 4.5.1; Velpotuzumab (Pola): lyophilized powder, prepared with normal saline before use.

[0154] Table 8 Grouping and dosing regimen of LY-24-0398PDX (TP53 mutation) model mice

[0155] Table 9 R-CHP dosage regimen

[0156] 4.5.5 LY-24-0004PDX (non-GCB, TP53 mutation) mouse model

[0157] Immunohistochemistry results of the model mice compared to patients showed the following: tumor cell LCA (+), CD79α (+), CD20 (+), CD43 (+), MUM-1 (+), Bcl-2 (+), Bcl-6 (- / +), Ki-67 (80%+), CD3 (-), CD5 (-), CD10 (-), CyclinD1 (-), CD23 (-), and AE1 / AE3 (-). NGS results detected mutations in the TP53, KMT2D, and PIM1 genes, with the TP53 mutation site being R248Q.

[0158] The pre-inoculation treatment, test indicators, killing and dissecting mice, and data processing for large-scale inoculation are the same as those in Section 4.5.1.

[0159] Grouped administration: mice with tumors reaching a volume of 100-200 mm 3 Grouped drug administration: This experiment still used Pola+R-CHP as a positive control. The mice were divided into 4 groups, namely, Control group, PM 5 mg / kg group, PM 10 mg / kg group and Pola+R-CHP group, with 5 mice in each group. The specific grouping and drug administration schedule are shown in Table 10.

[0160] Drug preparation: PM, rituximab, cyclophosphamide, doxorubicin, and prednisolone were prepared as before.

[0161] Table 10 LY-24-0004PDX (TP53 mutation) model mouse grouping and dosing regimen

[0162] 4.5.6 LY-24-0236PDX (GCB, TP53 mutation) mouse model

[0163] Immunohistochemistry results of the model mice were similar to those of the patients: tumor cells showed CD20 (+), CD79α (+), Bcl-6 (+), Bcl-2 (90%+), c-myc (40%+), MUM-1 (+), CD19 (+), CD30 (scattered+), Ki67 (70%+), CD22 (-), CD3 (-), CD5 (-), CD10 (-), Cyclin D1 (-), CD21 (-), and CD23 (-); in situ hybridization for EBER (-). NGS detected mutations in TP53, CCND3, PCLO, HIST1H1C, TNFRSF14, PRDM1, TMSB4X, LYN, and SOCS1 genes, with the TP53 mutation site being P300L.

[0164] The pre-inoculation treatment, test indicators, killing and dissecting mice, and data processing for large-scale inoculation are the same as those in Section 4.5.1.

[0165] Grouping: The mice were randomly divided into 6 groups, namely, Control group, PM 5 mg / kg group, PM 10 mg / kg group, Chidamide (Chi) 12.5 mg / kg group, PM 5 mg / kg + R-CHP group, and Pola + R-CHP group, with 6 mice in each group; the specific dosing schedule is shown in Table 11.

[0166] Drug preparation: PM, cedamide, rituximab, cyclophosphamide, vincristine, and prednisolone were prepared as before.

[0167] Table 11 LY-24-0236PDX (TP53 mutation) model mouse grouping and dosing regimen

[0168] 4.6 In vivo experiments in a xenograft mouse model of the DLBCL cell line U2932 (ABC, TP53 mutation)

[0169] DLBCL cell line U2932 cells in the logarithmic growth phase, the cell concentration was adjusted to 1×10 7 After the cells were collected, the cell suspension was stored on ice. 100 μL of U2932 cell suspension was drawn up with a 1 mL disposable sterile syringe and injected subcutaneously under the right armpit of the mouse to establish a U2932 xenograft mouse model. When the average tumor volume of the tumor-bearing mice reached 400-500 mm 3 The mice were divided into 6 groups at 4 hr. in total, including Control group, PM 5 mg / kg group, PM 10 mg / kg group, PM 5 mg / kg + RTX 10 mg / kg group, R-CHOP group, and PM + R-CHOP group, with 6 mice in each group. The specific grouping and dosing schedule are shown in Table 12.

[0170] Table 12 Grouping and dosing regimen of TP53 mutant U2932 cell line mouse model

[0171] 4.7 RNA-Seq Analysis of Tumor Tissues in LY-24-0179 and LY-24-0019 DLBCL PDX Models

[0172] The LY-24-0179(DEL)PDX model was treated in the same manner as the LY-24-0019(DHL)PDX model, and the tumor inoculation method was the same as before.

[0173] (1) Group administration: When the tumor volume of mice reached approximately 500 mm 3At 4 hr, mice were divided into two groups: the Vehicle group and the PM 5 mg / kg group, with 3 mice in each group. PM was prepared with normal saline and administered intravenously, while the Vehicle group received a blank formulation without PM, administered intravenously at the same volume as the PM group.

[0174] (2) Tumor tissue collection: 24 hours after a single dose, mice in each group were killed by cervical dislocation, and the tumor tissues were removed, snap-frozen in liquid nitrogen, and temporarily stored in a -80°C freezer. Tumor tissue samples (delivered on dry ice) were then sent to Shanghai Ouyi Biotechnology Co., Ltd. for transcriptome sequencing.

[0175] (3) Result analysis: RNA-Seq results were analyzed using R language, differential gene analysis was performed using the DESeq2 package, GSVA analysis was performed using the GSVA package, GO and GSEA enrichment analysis were performed using the cluster Profile package, and heat maps were drawn using the ComplexHeatmap package.

[0176] 4.8 Western blot analysis of apoptosis-related proteins and lymphoma poor prognosis-related proteins

[0177] Collect U2932, SU-DHL-4 and DOHH2 cells in the logarithmic growth phase, centrifuge at 1000 rpm / min for 3 min, discard the supernatant, resuspend the cells, and adjust the cell density to 2×10 6 2×10 cells / mL were plated per well of a 6-well plate. 6 Cells (1 mL) were plated and 1 mL of fresh culture medium prepared at different drug concentrations was added. The cells were incubated at 37°C for 24 hours. After 24 hours of drug exposure, the cells were harvested, centrifuged at 1500 rpm for 3 minutes, washed twice with pre-chilled PBS, and centrifuged at 3500 rpm for 3 minutes. The supernatant was discarded. Lysis buffer was prepared (RIPA lysis buffer, PMSF, and phosphatase inhibitor cocktail at a ratio of 100:1:1). 100-150 μL of the prepared lysis buffer was added to each sample. The sample was vortexed for 1 minute and placed on ice for 5 minutes. This process was repeated for a total of 30 minutes. To ensure complete protein lysis, the sample was sonicated three times, each for 1 minute, with 1 minute intervals. The sample was then centrifuged at 13,300 rpm at 4°C for 15 minutes. The supernatant was collected and placed in a pre-labeled 1.5 mL EP tube. The samples were temporarily placed on ice. Protein quantification was performed according to the protein quantification kit instructions. Finally, add 5× SDS-PAGE Loading Buffer to each sample, mix thoroughly, and incubate in a metal bath at 100°C for 10 minutes. Cool the samples to room temperature and store at -20°C.

[0178] After the completion of the experiment with LY-24-0206 PDX model mice, the mice were sacrificed, and the tumor tissues were removed and stored at -80°C. Since some of the LY-24-0179 and LY-24-0019 PDX model mice showed complete tumor regression, they were regrouped into the control group, the PM 5 mg / kg group, the PM 5 mg / kg combined with rituximab group, and the Chi (chidamide) 12.5 mg / kg group. Twenty-four hours after a single dose, the mice were sacrificed, and the tumor tissues were removed and stored at -80°C. Tumor tissue from each group was removed, weighed to 50-60 mg, cut into small pieces, and ground in a pre-chilled grinder using steel balls. Each sample was added with 500 μL of the prepared lysis buffer (prepared as above), and the samples were vortexed for 1 minute, kept on ice for 5 minutes, and the vortexing and on-ice lysis cycles were repeated for a total of 30 minutes. The samples were then sonicated three times for 1 minute, with 3 minutes between each. Centrifuge at 13,300 rpm / min at 4°C for 15 minutes, remove the supernatant, and place on ice until ready for use. Measure absorbance at 595 nM using a microplate reader and calculate the protein concentration of each sample. Finally, perform protein denaturation. Follow standard Western blotting procedures to detect changes in protein expression levels.

[0179] 4.9 Flow cytometry detection of tumor-infiltrating T cell subtypes in model mice

[0180] 4.9.1 Establishment of the A20 Subcutaneous Tumor Model and Detection of Tumor-Infiltrating T Cell Subtypes

[0181] A20 cells in the logarithmic growth phase were collected, centrifuged, the supernatant was discarded, and washed twice with sterile PBS to adjust the cell density to 5×10 7 / mL (subcutaneous inoculation). Mix the prepared cell suspension thoroughly and take 100 μL (5×10 6 The cell suspension was inoculated subcutaneously in the right armpit of the mouse. After inoculation, the mice were closely monitored for leakage at the inoculation site and for any abnormalities in the skin. When the average tumor volume of the tumor-bearing mice reached 200 mm 3 The mice were divided into four groups: Vehicle, PM 10 mg / kg, and R-CHOP, with six mice in each group. Dosing was the same as before. The Vehicle group received a blank formulation without PM, administered intravenously at the same volume as the PM group. On day 14 of dosing, the mice in the Vehicle and PM 10 mg / kg groups were sacrificed by cervical dislocation, and tumor tissue was removed. Single-cell suspensions were prepared and flow cytometry was performed.

[0182] (1) Preparation of digestion solution: First, add 5 mL of PBS to 100 mg of papain, 50 mg of collagenase I, 50 mg of collagenase IV, and 100 mg of DNA enzyme, and then add 3 mL of PBS to 30 mg of hyaluronidase. Then, take 500 μL of the above-prepared collagenase I, 500 μL of collagenase IV, 200 μL of papain, 200 μL of hyaluronidase, and 25 μL of DNA enzyme, mix them together, and dilute to 50 mL with HBSS solution for later use.

[0183] (2) Preparation process for single cell isolation: mince the tumor tissue and transfer it to a 2 mL EP tube. Add 500 μL of the above-prepared digestion solution to each tube. Digest at 37°C, 250 rpm / min for 15-20 min. Filter with a membrane. Add 2 mL of culture medium containing fetal bovine serum to neutralize the digestion. Operate on ice. Add red blood cell lysis buffer to lyse the red blood cells. Centrifuge at 500 g for 5 min. Discard the supernatant and resuspend the cells in PBS.

[0184] (3) Detection of tumor-infiltrating lymphocytes by flow cytometry: Add 1.5 μL of mouse CD3, CD4, CD8, CD62L, and CD44 antibodies to the sample resuspended in PBS, avoid staining for 15 min, centrifuge at 500 g for 5 min, resuspend the cells in 300 μL of PBS, filter through a 300-mesh filter, and then detect on the flow cytometer.

[0185] 4.10 Flow cytometry detection of major histocompatibility complex (MHC) I and MHC II in DLBCL cells

[0186] Take U2932 cells in the logarithmic growth phase, count the cells, resuspend the cells in complete medium, and adjust the cell density. Use 6-well plates for plating, 5×10 cells per well. 5 Cells were treated with various concentrations of PM and chidamide, with control groups set up at PM concentrations of 0.5, 1, and 2.5 nM; and chidamide concentrations of 100, 500, and 1500 nM. Culture was continued in an incubator. After 24 hours, cells were harvested, washed twice with PBS, centrifuged at 500 g for 5 minutes, and the supernatant discarded. Cells were resuspended in PBS and incubated in the dark for 15 minutes with 1.5 μL of human MHC-I and MHC-II antibodies. The cells were washed once with PBS and finally resuspended in 400 μL of PBS. The cells were filtered through a 300-mesh filter before analysis.

[0187] 5. Statistical Analysis Used in Examples

[0188] Data and statistical analysis were performed using Adobe Illustrator 2021, Graphpad Prism 9.0, R software 4.2.1, FlowJo 10.6.2, and ModFit LT 3.1. FlowJo software was used for analysis, organization, and quantification of apoptosis flow cytometry results; ModFit LT software was used for analysis, organization, and quantification of cell cycle flow cytometry results. Comparisons between two groups were performed using the t-test, and comparisons between multiple groups were performed using one-way analysis of variance. Survival curves were analyzed using the log-rank test, with P < 0.05 considered statistically significant. Each experiment was repeated three times, and data are presented as mean ± standard deviation.

[0189] Example 1 HDAC class I and class IIb are highly expressed in DLBCL patients

[0190] Abnormal expression of HDACs is closely associated with a variety of hematological tumors and solid tumors. First, the expression of HDACs in normal human peripheral blood leukocytes and newly diagnosed DLBCL tumor tissues was analyzed through public databases. By analyzing the RNA-Seq results of tumor tissues of 47 DLBCL patients in the TCGA database and 444 normal human peripheral blood leukocytes in the GTEx database, it was found that compared with peripheral blood leukocytes, newly diagnosed DLBCL patients showed significantly higher expression of HDAC class I (HDAC 1, HDAC 2, HDAC 3 and HDAC 8), HDAC class IIb (HDAC 6, HDAC 10), HDAC 7, HDAC 9 in HDAC class IIa, and HDAC class IV (HDAC 11) (Figure 1).

[0191] Although HDAC 7, HDAC 9, and HDAC 11 within class IIa are also overexpressed, pan-HDAC inhibitors have greater toxic side effects. For example, in a clinical trial of panobinostat for the treatment of relapsed / refractory multiple myeloma, adverse events led to one-sixth of patients discontinuing panobinostat and requiring hospitalization. Toxicity affected vital organs such as the hematology, heart, gastrointestinal tract, liver, and kidneys, and the proportion of patients dying from causes unrelated to multiple myeloma was as high as 6.8%. These severe, even fatal, side effects may be related to the inhibition of class IIa and IV HDACs. Furthermore, myeloid-derived suppressor cells (MDSCs) are a group of cells with potent immunosuppressive functions. MDSCs have been observed to be significantly increased in B-cell lymphomas (including Hodgkin lymphoma, DLBCL, and follicular lymphoma) and are closely associated with poor prognosis and shortened overall survival. Because MDSCs are a crucial component of the immunosuppressive tumor microenvironment, they can inhibit various T cell functions within the tumor microenvironment, allowing tumors to evade immune surveillance and thus promote tumor growth. Studies have shown that HDAC11-deficient mice have increased MDSCs and enhanced immunosuppressive effects. T cell lymphomas transplanted into HDAC11-deficient mice are more aggressive than those in HDAC11 wild-type mice, suggesting that HDAC11 inhibits MDSCs. Furthermore, Treg cells are also anti-tumor immunosuppressive agents, and data also indicate that inhibition of HDAC9 or HDAC class III sirtuin-1 can enhance Treg cell suppressive activity. Therefore, considering the potential for toxicity and tumor immunosuppression, highly selective HDAC I / IIb inhibitors are more suitable for the treatment of DLBCL patients than pan-HDAC inhibitors. The overexpression of HDAC class I and IIb in DLBCL patients provides a rationale for the use of highly selective HDAC I / IIb inhibitors (PM) for the treatment of DLBCL.

[0192] Example 2 Epigenetic and TP53-related gene mutation analysis in DLBCL patients

[0193] Epigenetic abnormalities are closely associated with drug resistance and recurrence in DLBCL. Studies have shown that drug resistance in DLBCL is associated with tumor heterogeneity, the tumor microenvironment, and host factors. Abnormalities in epigenetic regulatory genes (such as EZH2, CREBBP, MEF2B, KMT2C, and KMT2D) are a key factor in tumor heterogeneity. Furthermore, mutations in genes involved in epigenetic regulation are common in DLBCL, including histone methyltransferases KMT2C, KMT2D, and EZH2; histone acetyltransferases CREBBP, EP300, and IRF4; chromosome remodeling genes HIST1H1E and ARID1A; and the DNA methylation gene TET2. We used the cBioportal database to analyze the gene mutations in 1295 newly diagnosed DLBCL patients from five research centers. (Figure 2A) These mutations confirm the prevalence of these genes in DLBCL, with KMT2D mutations occurring in 25% of patients and CREBBP mutations in 13%.

[0194] Professor Shi Yuankai's team (Jiang S, Qin Y, Jiang H, Liu B, Shi J, Meng F, Liu P, Yang J, Yang S, He X, Zhou S, Gui L, Liu H, Lin J, Han-Zhang H, Shi Y: Molecular profiling of Chinese R-CHOP treated DLBCL patients: Identifying a high-risk subgroup. International journal of cancer 2020, 147: 2611-20.) included 105 patients with non-primary central nervous system DLBCL who received first-line R-CHOP regimen and had baseline tissue samples for baseline mutation spectrum analysis and found that high-frequency mutations in DLBCL patients included PIM1 (33%), MYD88 (29%), BCL-2 (29%), TP53 (29%), CD79B (25%) and KMT2D (24%). Furthermore, the primary refractory rate among patients with TP53 mutations was 87.0% (20 / 23), while among patients with TP53 wild-type disease, the primary refractory rate was only 50.0% (29 / 58, P = 0.009). Among patients carrying disruptive TP53 mutations, the primary refractory rate was as high as 91.7%, indicating that patients with TP53 mutations are more likely to be primary refractory and have a worse overall survival. Using the same approach described above, we analyzed the gene mutation profile of 1,295 newly diagnosed DLBCL patients from five research centers using the cBioportal database and found that TP53, MYD88, and PIM1 mutations were found in 12%, 17%, and 16%, respectively (Figure 2B), making them very common mutations in DLBCL and associated with a poor prognosis.

[0195] Example 3 In vitro antitumor activity of PM in the treatment of DLBCL including DEL lymphoma and TP53 deletion and mutation

[0196] 3.1 PM significantly inhibits the proliferation of DLBCL cell lines harboring multiple poor prognosis-related gene mutations

[0197] As previously mentioned, abnormalities in TP53, c-MYC, MYD88, BCL-2, and CD79B are important genes associated with poor prognosis in DLBCL. Therefore, we investigated the in vitro antiproliferative activity of PM in seven human DLBCL cell lines harboring these genetic abnormalities: U2932 (ABC subtype, TP53 mutation, and BCL-2 rearrangement), SU-DHL-4, SU-DHL-6, and Karpas 422 (GCB subtype, TP53 mutation, and BCL-2 rearrangement), DOHH2 (GCB subtype, c-MYC and BCL-2 rearrangement), OCI-LY18 (GCB subtype, TP53 mutation, c-MYC and BCL-2 rearrangement), and HBL-1 (ABC subtype, TP53, MYD88, and CD79B mutations). Different concentrations of PM were treated with these cell lines for 48 and 72 hours, yielding IC50 values ​​below 2.5 nM. Consistent with PM's results on HDACI / IIb enzyme inhibition, under the same conditions, PM exhibited significantly superior antiproliferative activity compared to panobinostat (LBH589) and chidamide, exceeding chidamide by more than 500-fold (Figures 3, 4, and Table 13). PM demonstrated excellent antiproliferative activity across GCB subtypes (SU-DHL-4, SU-DHL-6, Karpas422, DOHH2, OCI-LY18), ABC subtypes (U2932, HBL-1), and high-grade DLBCL with a poor prognosis or those harboring prognostic mutations. Therefore, PM exhibited excellent in vitro antiproliferative activity against a variety of human DLBCL cell lines harboring multiple prognostic mutations and high-grade DLBCL subtypes.

[0198] Table 13 IC50 values ​​of antiproliferative activity of PM, chidamide and LBH589 against 7 human DLBCL cell lines *vs.PM, P<0.05; **vs.PM, P<0.01; ***vs.PM, P<0.001; ****vs.PM, P<0.0001.

[0199] 3.2 PM arrests DLBCL cell cycle at G0 / G1 phase and is significantly superior to chidamide

[0200] From the seven human DLBCL cell lines mentioned above, three highly malignant cell lines were selected for cell cycle analysis: SU-DHL-4 (DLBCL NOS, GCB subtype), U2932 (TP53 mutation, ABC subtype), and DOHH2 (DHL, GCB subtype). These three cell lines were treated with varying concentrations of PM and chidamide for 24 hours, and then the cell cycle was assessed by flow cytometry. The results showed that in SU-DHL-4 cells, the proportion of cells in the G0 / G1 phase significantly increased with increasing PM concentration. At 2.5 nM PM, the proportion of cells in the G0 / G1 phase increased from 43.07% in the control group to 87.90%. Compared to chidamide (500, 1500, and 4500 nM), PM exhibited a highly potent cytostatic effect at lower concentrations (0.5, 1, and 2.5 nM). Similar results were observed in U2932 and DOHH2 cells (Figure 5). The above results indicate that PM blocks the DLBCL cell cycle at the G0 / G1 phase and is more than 1000 times more effective than chidamide.

[0201] 3.3 PM induces apoptosis in a variety of human DLBCL cells, including double-hit and TP53 mutant cells, in a concentration-dependent manner and is superior to chidamide

[0202] Subsequently, this study also examined apoptosis in the three aforementioned DLBCL cell lines. SU-DHL-4, U2932, and DOHH2 cells were treated with varying concentrations of PM and chidamide for 48 hours, and apoptosis was assessed by flow cytometry. As shown in Figures 6, 7, and 8, PM induced apoptosis in these cells in a concentration-dependent manner. At a concentration of 10 nM, PM elicited an apoptotic rate of 80-90%, while at a concentration of 2000 nM, chidamide elicited only a 29-33% apoptotic rate. Furthermore, with increasing PM concentration, protein expression of the pro-apoptotic proteins cleaved caspase 3, cleaved caspase 9, and cleaved PARP was significantly upregulated. At equivalent concentrations, PM elicited a more pronounced upregulation than chidamide. These results demonstrate that PM induces apoptosis in DLBCL cells, with superior activity compared to chidamide.

[0203] Example 4 In vivo anti-tumor effects of PM in treating various DLBCL PDX model mice

[0204] In vitro studies confirmed that PM exhibited superior antitumor activity against various subtypes and high-grade DLBCL. The in vivo antitumor activity of PM was further explored using DLBCL PDX models. Three DLBCL PDX mouse models were established: LY-24-0206 (DLBCL NOS, GCB subtype), LY-24-0179 (DEL, non-GCB subtype), and LY-24-0019 (DHL, GCB subtype). After tumors developed in the DLBCL PDX model mice, tumor tissue was obtained for pathology, immunohistochemistry, and FISH analysis. The results were consistent with those from the corresponding patients, confirming the successful establishment of the PDX models.

[0205] 4.1 PM is more effective than the first-line treatment regimen R-CHOP and the new drug Selinexor in the DLBCL PDX (NOS, GCB) mouse model

[0206] A GCB-type DLBCL (NOS, LY-24-0206) PDX mouse model was established. When the tumor volume reached 200-300 mm 3 The patients were divided into groups. They were Control group, PM 5mg / kg group, PM 10mg / kg group, PM 5mg / kg+RTX 10mg / kg group, and PM 5mg / kg+R-CHOP group, and the first-line treatment regimen R-CHOP group was used as a positive control. In addition, the overall response rate (ORR) of XPO1 inhibitor Selinexor monotherapy for relapsed / refractory DLBCL was 28%, the complete response (CR) rate was 12%, the median follow-up time was 11.1 months, the median duration of response was 9.3 months, and the median OS was 9.1 months. It has been approved for the treatment of relapsed / refractory DLBCL. We also compared the efficacy of PM and Selinexor on this PDX model. The specific dosing regimens are shown in Tables 2 and 3.

[0207] Results showed that all treatment groups significantly inhibited tumor growth compared to the control group. Treatment ended on day 16, and mice were sacrificed. Tumors in each group were weighed. Tumor inhibition rates were 91.10%, 95.42%, 99.60%, 75.99%, 100.00%, and 73.54% for the PM 5mg / kg group, PM 10mg / kg group, PM 5mg / kg + RTX 10mg / kg group, PM 5mg / kg + R-CHOP, R-CHOP, and Selinexor 10mg / kg groups, respectively. Tumor inhibition rates for PM 10mg / kg alone and the PM 5mg / kg combination groups were significantly superior to those observed in the clinical treatment regimens RTX combined with CHOP and Selinexor 10mg / kg. In the PM 10mg / kg group, three mice (3 / 6) experienced complete tumor regression, while in the PM 5mg / kg combined with RTX group, four mice (4 / 6) experienced complete tumor regression. In the PM 5mg / kg combined with R-CHOP group, all mice experienced complete tumor regression. During treatment, mice in the R-CHOP group experienced significant weight loss, while mice in the selinexor group experienced diarrhea, and one mouse (1 / 6) died. The activity, diet, and weight of mice in the PM alone and PM combined with RTX groups remained normal, with no significant toxic side effects observed.

[0208] 4.2 PM monotherapy is more effective than R-CHOP and Selinexor in the dual-expression DLBCL PDX (non-GCB) mouse model

[0209] DEL DLBCL is characterized by strong invasiveness and low response rate to first-line treatment. Therefore, we further established a DEL PDX (non-GCB, LY-24-0179) mouse model to explore the in vivo anti-tumor activity of PM against DEL diffuse large B-cell lymphoma. 3 The patients were divided into 6 groups for drug administration at around 37 hr. The groups included Control group, PM 5 mg / kg group, PM 10 mg / kg group, PM 5 mg / kg + RTX 10 mg / kg group, R-CHOP group, and Selinexor 10 mg / kg group. The specific drug administration schedule is shown in Table 4.

[0210] The results, as shown in Figure 9A, show that compared to the control group, all treatment groups significantly inhibited tumor growth, with PM alone or in combination with RTX showing the most significant efficacy. After one week of treatment, tumors in mice gradually regressed. By day 9, complete tumor regression was observed in four mice in the PM 10 mg / kg group and in the PM 5 mg / kg combined with RTX group. At the end of treatment and subsequent sacrifice on day 21, complete tumor regression was observed in four of six mice in the PM 5 mg / kg alone group, five of six mice in the PM 10 mg / kg alone group, and all six mice in the PM 5 mg / kg combined with RTX group. Tumor inhibition rates for the PM 5 mg / kg, PM 10 mg / kg, and PM + RTX groups were 99.76%, 99.97%, and 100%, respectively, significantly superior to the 73.52% inhibition rate achieved with the first-line chemotherapy regimen R-CHOP and the 93.48% inhibition rate achieved with selinexor 10 mg / kg. Consistent with the results in the PDX model mice described above, mice in the PM alone and PM combined groups maintained normal diet, activity, and body weight during treatment, with no significant side effects observed. However, mice in the selinexor group experienced diarrhea, significant weight loss, and one mouse died (Figure 9B). These results demonstrate that PM alone and in combination with RTX exhibited robust antitumor activity in the DEL PDX model.

[0211] Since a multicenter phase III trial of chidamide combined with R-CHOP for the treatment of newly diagnosed DEL is underway and has achieved good results, we have established a DEL PDX (non-GCB, LY-24-0179) mouse model to compare the efficacy of PM+R-CHOP and Chi+R-CHOP (chidamide combined with R-CHOP). Because PM alone has shown significant anti-tumor activity in the first batch of experiments, with complete tumor regression in some mice, the tumor volume of the mice in this model reached 300-400mm. 3 The specific dosing schedule is shown in Table 5.

[0212] The results are shown in Figure 10A. Compared with the Chi+R-CHOP group, the tumor volume of mice in the PM+R-CHOP treatment group was significantly reduced at each time point, and the tumors of most mice disappeared after one week of treatment. During the treatment process, due to the presence of chemotherapy drugs, the weight of mice in both groups decreased to a certain extent (Figure 10B). When the mice were killed at the end of the drug administration on the 18th day, the tumors of 4 out of 5 mice in the PM+R-CHOP group completely disappeared, with a tumor inhibition rate of 99.88%, while the tumor inhibition rate of the Chi+R-CHOP group was 61.96%. The above results show that the efficacy of PM alone or in combination in the treatment of DEL PDX models is significantly better than the existing treatment options R-CHOP, Selinexor and Chi+R-CHOP.

[0213] 4.3 PM treatment resulted in complete tumor regression in the double-hit DLBCL PDX (GCB subtype) model mice

[0214] In addition to the above PDX models, we also explored the efficacy of PM in DHL PDX mouse models. A DHL (GCB subtype, LY-24-0019) PDX mouse model was established. When the tumor volume grew to 200 mm 3 Around 3 d, the patients were divided into groups for treatment, including control group, PM 5 mg / kg group, PM 10 mg / kg group, PM 5 mg / kg + RTX 10 mg / kg group, and R-CHOP group. The specific dosing schedule is shown in Table 5.

[0215] As shown in Figure 11A, compared with the Control group, each treatment group significantly inhibited the growth of tumors in the model mice at each time point during treatment. During the treatment process, the tumors of the mice in the R-CHOP group gradually increased, while the tumor volume of the PM alone group and the PM combined with RTX treatment group gradually decreased. On the 9th day of administration, the tumors of 3 mice in the PM 5mg / kg group were completely regressed, and the tumors of 4 mice in the PM 10mg / kg group and the PM 5mg / kg combined with RTX group were completely regressed. Treatment was terminated on the 18th day of administration. The tumors of 4 mice in the PM 5mg / kg group were completely regressed, and the tumors of all mice in the PM 10mg / kg group and the PM 5mg / kg combined with RTX group were completely regressed (Figure 11A). Tumors in mice were weighed across treatment groups. Tumor inhibition rates were 99.87%, 100%, and 100% in the PM 5mg / kg, PM 10mg / kg, and PM 5mg / kg + RTX 10mg / kg groups, respectively. These rates significantly outperformed the 71.99% inhibition rate achieved by the first-line five-drug combination, R-CHOP. Throughout treatment, mice in the PM and combination groups maintained normal body weights (Figure 11B), and no significant toxic side effects were observed.

[0216] Furthermore, the model was re-established, and the efficacy of low-dose PM 5mg / kg alone was compared with that of Selinexor 10mg / kg alone. After 16 days of treatment, the tumors of the five mice in the PM 5mg / kg alone group completely regressed, with a tumor inhibition rate of 100%, while the tumor inhibition rate of Selinexor was only 59.22%, and the body weight decreased by 13.37%. The mice also had diarrhea and had more serious toxic side effects (Figure 12). In summary, PM also exerted its excellent anti-tumor activity in the double-hit PDX (GCB subtype, LY-24-0019) mouse model, with efficacy significantly better than the first-line treatment regimen R-CHOP and the XPO1 inhibitor Selinexor.

[0217] 4.4 PM has no obvious visceral toxicity to DLBCL PDX model mice

[0218] Previous studies have shown that PM treatment has excellent efficacy in various DLBCL PDX models, with mice maintaining normal diet and activity, and no significant weight loss was observed. Furthermore, organ tissues collected from each treatment group in the DEL DLBCL (non-GCB, LY-24-0179) PDX mouse model were subjected to pathological evaluation using hematoxylin and eosin staining. Heart, liver, spleen, lung, kidney, stomach, and small intestine were obtained from three mice in each group for hematoxylin and eosin staining, and organ grading was assessed according to the International Norms for Terminology and Diagnostic Criteria in Mouse Pathology (INHAND) criteria (Table 14). Results showed that one mouse (1 / 3) in the PM 5 mg / kg group developed grade 1 intestinal epithelial cell desquamation, while one mouse (1 / 3) in each of the PM 10 mg / kg group and the PM 5 mg / kg + RTX group developed grade 2 intestinal epithelial cell desquamation. No significant abnormalities were observed in other organs. In the R-CHOP group, one mouse (1 / 3) developed grade 1 cardiac degeneration, and one mouse (1 / 3) developed grade 3 renal tubular degeneration. In the selinexor group, two mice (2 / 3) experienced grade 3 intestinal epithelial cell exfoliation (Figure 13). This suggests that, consistent with the weight changes during treatment, PM had no significant toxic side effects on the major organs of DLBCL PDX model mice, and its toxicity was lower than that of R-CHOP and selinexor.

[0219] Table 14 Evaluation criteria for pathological changes in mouse organs

[0220] Example 5 Exploration of the mechanism of action of PM in the treatment of DLBCL

[0221] In vitro and in vivo experiments confirmed that PM has significant anti-tumor activity against DLBCL. It not only has excellent in vitro and in vivo efficacy against DLBCL NOS, but is also sensitive to DEL and DHL with poor prognosis. Next, the mechanism will be explored at both the protein level and the transcriptome level. DEL (non-GCB subtype, LY-24-0179) and DHL (GCB subtype, LY-24-0019PDX) PDX models were established. When the average tumor volume was 500-1000mm 3 The mice were divided into vehicle and PM 5mg / kg groups, with three mice in each group. Twenty-four hours after dosing, tumor tissues were harvested for RNA-Seq analysis and bioinformatics analysis. Furthermore, the key sequencing results were validated at the protein level. PM-treated DLBCL cell lines and mouse models were analyzed using western blot and flow cytometry to examine key protein expression and changes in related signaling pathways.

[0222] 5.1 PM significantly downregulates the poor prognosis biomarker c-MYC and its gene set

[0223] C-Myc, an oncogene located on chromosome 8q24, is one of the most important biomarkers of poor prognosis in DLBCL. A systematic review and meta-analysis of 24 studies, including 4,662 patients with DLBCL, showed that abnormalities in C-Myc (including gene abnormalities and elevated expression at both mRNA and protein levels) were strongly associated with poor overall survival. Furthermore, numerous studies have also confirmed that abnormalities in C-Myc are associated with shorter survival.

[0224] GSEA (Hallmark gene set) enrichment analysis was performed on RNA-Seq results from tumor tissues of DEL (non-GCB subtype, LY-24-0179) and DHL (GCB subtype, LY-24-0019 PDX) PDX models 24 hours after a single dose of vehicle and PM 5 mg / kg. As shown in Figure 14, PM treatment significantly enriched the Myc downregulated target gene sets "MYC_TARGETS_V1" and "MYC_TARGETS_V2" in both PDX models (P < 0.01).

[0225] Furthermore, changes in c-MYC protein expression levels were detected in DLBCL cell lines and PDX model mice treated with PM. WB detection of human DLBCL cell lines revealed that in DLBCL cell lines SU-DHL-4, U2932, and DOHH2, PM concentration-dependently downregulated c-MYC protein expression levels in cell lines (Figure 15A). In addition, Myc can also regulate cell differentiation and proliferation through transcriptional amplification of its downstream target genes. MCL-1 protein is involved in controlling cell apoptosis, making tumor cells resistant to chemotherapy drugs. Similarly, PM also concentration-dependently downregulated MCL-1 protein expression levels in cell lines. Next, we also performed WB detection on tumor tissues of different dosing groups of three DLBCL PDX model mice treated with PM, all showing that PM significantly downregulated the expression of c-MYC at the protein level (Figure 15B). Moreover, consistent with the in vivo efficacy results, PM's ability to inhibit c-MYC expression was better than that of the R-CHOP, Selinexor, and Chidamide treatment groups. Therefore, PM can significantly inhibit the MYC gene set and c-MYC transcription and protein levels in DLBCL tumor tissues, thereby affecting the growth and proliferation of DLBCL tumor cells. The results of this study provide an important basis for PM treatment of DLBCL with high expression of c-MYC protein levels.

[0226] 5.2 PM treatment significantly downregulated the expression of key gene sets associated with tumor survival in PDX model mice

[0227] Previous RNA-seq sequencing analysis of PM-treated multiple myeloma model mice showed that PM treatment inhibited the key gene sets involved in the survival of various tumor cells. Therefore, GSVA and GSEA analyses of the Hallmark gene set were performed based on the sequencing results of the DEL (non-GCB subtype, LY-24-0179) and DHL (GCB subtype, LY-24-0019PDX) PDX mouse models.

[0228] The results showed that, consistent with previous studies, in addition to downregulating MYC target genes, PM treatment also significantly enriched and downregulated key gene sets associated with DLBCL tumor cell survival, including DNA_REPAIR, E2F_TARGETS, G2M_CHECKPOINT, MTORC1_SIGNALING, OXIDATIVE_PHOSPHORYLATION, and UNFLOD_PROTIEN_RESPONSE (Figures 16, 17, 18, and 19). Literature reports indicate that pan-HDAC inhibitors (including vorinostat) are hyposensitive to DLBCL subtypes of oxidative phosphorylation metabolism and exhibit poor therapeutic efficacy. This study, based on Hallmark gene set enrichment analysis, also showed that PM significantly downregulated genes related to oxidative phosphorylation. These results further confirm that PM treatment exhibits excellent anti-tumor activity in DLBCL.

[0229] 5.3 PM treatment upregulates T cell activation and proliferation-related gene sets in tumor tissues of DLBCL model mice

[0230] Similarly, GSVA analysis based on the Hallmark gene set showed that after 24 hours of PM treatment at 5 mg / kg, the gene sets TNFA_SIGNALING_VIA_NFKB, INFLAMMATORY_RESPONSE, IL2_STAT5_SIGNALIN, HYPOXIA, COMPLEMENT, and INTERFERON_GAMMA_RESPONSE related to interferon response, inflammation, and immune response were significantly enriched and upregulated in the tumor tissues of two PDX model mice (Figures 19 and 20). GSEA enrichment analysis also showed that the upregulated genes were mainly enriched in the above gene sets (Figures 20 and 21), suggesting that PM may affect immunity in vivo.

[0231] We then performed GSEA analysis based on the GO gene set and found that the upregulated genes were mainly enriched in gene sets related to T cell activation, T cell proliferation, T cell co-stimulation, T cell differentiation, and positive regulatory T cell activation and positive regulatory T cell-mediated cytotoxicity (Figures 22 and 23). These results suggest that after PM treatment, genes related to regulating T cell activation, proliferation, and differentiation are upregulated, but whether PM has an effect on T cell activation and proliferation requires further verification.

[0232] 5.4 PM treatment significantly increased the proportion of CD8+ tumor-infiltrating T cells and effector / memory T cells

[0233] Previous sequencing results from mouse models have shown that PM treatment may promote T cell proliferation and activation in vivo. Therefore, we established a DLBCL mouse model with a normal immune system by inoculating the A20 cell line (a murine B-cell lymphoma cell line) into Balb / c mice to explore the effects of PM treatment on T cell proliferation and activation.

[0234] After the tumor was formed in mice, when the tumor volume grew to 200mm 3 The mice were divided into vehicle, PM 10 mg / kg, and R-CHOP groups. After two weeks of continuous treatment, PM significantly inhibited tumor growth. Tumor tissue was weighed, and the results showed that the tumor inhibition rates in the PM 10 mg / kg and R-CHOP treatment groups were 60.13% and 34.71%, respectively (Figures 24A and B). Furthermore, the mouse tumor tissues were isolated for single cells, and T cell infiltration was analyzed by flow cytometry. The results showed that, consistent with PM treatment of multiple myeloma 5TMM model mice, the proportion of CD8+ tumor-infiltrating lymphocytes to CD3+ T cells in the tumor tissues of A20 model mice in the PM 10 mg / kg group was significantly increased compared with the vehicle group (PM vs vehicle, 34.97% vs 20.40%). The proportion of CD3+CD8+CD44+CD62L- effector memory T cells was also significantly increased (PM vs vehicle, 85.78% vs 69.48%) (Figures 24C-F). In summary, PM treatment significantly promoted the proliferation and activation of CD8+ T cells in tumor tissues of model mice.

[0235] 5.5 PM upregulates MHC-I and MHC-II expression in DLBCL tumor cells, enhancing antigen presentation

[0236] Numerous studies have reported on the effects of HDAC inhibitors on tumor immunity, with the most extensively studied finding being their ability to enhance tumor immunogenicity. To reduce T cell recognition and achieve immune evasion during lymphomagenesis, B-cell lymphomas often downregulate the expression of MHC class I and II molecules on the tumor cell surface. It has been reported that over 50% of patients with DLBCL exhibit loss of MHC class I expression. Reduced MHC class II expression is not only common in DLBCL but also occurs in other types of mature B-cell lymphomas. Downregulation or loss of MHC class I and II reduces tumor immunogenicity, decreases the percentage of CD8 and CD4 tumor-infiltrating lymphocytes (TILs), induces resistance to immunotherapy, and is associated with poor prognosis and survival. In various tumor types, both selective class I and pan-HDAC inhibitors have been shown to increase the expression of genes involved in the MHC class I antigen presentation pathway and promote the expression of MHC class I molecules on the tumor cell surface, leading to tumor cell recognition and killing by cytotoxic CD8+ T cells. Secondly, HDAC inhibitors can also promote the MHC class II antigen processing and presentation pathway, leading to the activation of CD4+ T cells.

[0237] Further analysis of the RNA-Seq results above revealed that, in both the LY-24-0179 (DEL) and LY-24-0019 (DHL) models, the PM 5 mg / kg group showed upregulation of MHC class I and class II genes, including HLA-A, HLA-B, HLA-C, as well as HLA-DP, HLA-DQ, and HLA-DR, in tumor tissue compared to the vehicle group (Figure 25). Furthermore, upregulation of MHC protein complexes and protein complex binding-associated genes, such as HLA-DMA, HLA-DMB, HLA-DOA, HLA-DOB, CD4, CD74, CD8A, ATP1B1, LAG3, LILRB1, and LILRB2, was also observed (Figures 26 and 27). Therefore, we speculate that PM may upregulate the expression of MHC class I and class II on the surface of DLBCL tumors.

[0238] For further verification, we treated U2932 cells with different concentrations of PM and chidamide, and detected the expression of MHC on the surface of U2932 cells 24 hours later. The results are shown in Figure 28. With the increase of PM concentration, the expression of MHC-I and MHC-II gradually increased, and PM at around 0.5 nM can lead to a significant upregulation of MHC-I and MHC-II expression, while chidamide only caused a significant upregulation of MHC I at 1500 nM, and 500 nM caused a significant upregulation of MHC-II expression.

[0239] The above data show that PM can upregulate the expression of MHC-I and MHC-II on the surface of DLBCL cells, and its upregulation ability is significantly better than that of cedabendine, thereby enhancing tumor cell antigen presentation, helping T cell recognition, and avoiding immune escape.

[0240] Example 6 Efficacy and Mechanism of PM in Treating TP53-Deficient and Mutated DLBCL and TP53-Mutated DLBCL with Dual Expression

[0241] 6.1 PM has excellent in vitro antitumor activity against TP53 mutant or null DLBCL cell lines and downregulates the level of P53 mutant protein

[0242] TP53 mutation is also a significant factor in poor prognosis in DLBCL. Therefore, a literature search was conducted on the seven human DLBCL cell lines used in the present invention. It was found that U2932, SU-DHL-4, SU-DHL-6, Karpas 422, OCI-LY18, and HBL-1 all harbor TP53 deletions or mutations, while DOHH2 harbors wild-type TP53. As shown in Figure 3, PM remains sensitive to DLBCL cell lines harboring TP53 mutations or deletions, with a 72-hour IC50 value of less than 2.5 nM. Furthermore, it was found that PM also exhibited concentration-dependent pro-apoptotic and G0 / G1 cell cycle arrest in the TP53 mutation- or deletion-resistant cell lines U2932 and SU-DHL-4. These data demonstrate that PM exhibits excellent in vitro anti-tumor activity against DLBCL cells harboring TP53 mutations or deletions.

[0243] Since P53 primarily affects its localization and activity through post-translational modifications, acetylation is a key factor in P53 activation. HDAC inhibitors promote histone acetylation and also increase non-histone acetylation levels. Existing reports indicate that inhibition of HDAC 1, HDAC 2, HDAC 3, HDAC 6, and HDAC 8, particularly HDAC 6 and HDAC 8, is the primary driver of elevated acetylated P53 expression. Knockout of HDAC 8 also reduces HOXA 5 expression, further blocking HOXA 5-activating mutant TP53 transcription. Therefore, the TP53 mutant cell lines SU-DHL-4 and U2932 were treated with varying concentrations of PM, and the effects of PM on acetylation levels of histones H3 and H4, as well as non-histone P53, were examined by Western blot. The results showed that PM upregulated acetylation of histones H3 and H4. Furthermore, with increasing PM concentrations, the expression level of mutant P53 gradually decreased, while the expression level of acetylated P53 gradually increased (Figure 29). The results of this study showed that PM downregulates the expression of mutant P53 and upregulates the expression of acetylated P53, exerting its tumor suppressor function.

[0244] 6.2 PM is significantly more effective than Pola-R-CHP and Venetoclax-R-CHOP in treating TP53 (R248Q)-mutated DLBCL (non-GCB) PDX

[0245] Patients with TP53-mutated DLBCL have a poor prognosis, and those with TP53-mutated and dual-expressing DLBCL have lower remission rates and shorter survival. In vitro experiments have demonstrated that PM also exhibits excellent anti-proliferative, pro-apoptotic, and cell cycle arrest activities against DLBCL cell lines with TP53 mutations or deletions, as well as a DLBCL cell line with TP53 mutations and dual expression (U2932). Furthermore, we constructed PDX models of DLBCL with TP53 deletions and multiple mutations. We used the TP53-mutated U2932 cell line to establish xenograft mouse models, as well as PDX models with TP53 mutations and dual expression, to explore the in vivo efficacy of PM in mice with TP53-deficient and mutant DLBCL, as well as TP53-mutated and dual-expressing DLBCL models.

[0246] The LY-24-0004PDX model has a variety of common DLBCL gene mutations with poor prognosis, including TP53 (mutation site: R248Q), KMT2D, and PIM1. Since multiple PDX models have previously confirmed that PM is more effective than the R-CHOP regimen, and the Pola+R-CHP regimen has been approved as the first-line treatment for DLBCL, based on the above two reasons, the R-CHOP group was not set up for this grouping, but the Pola+R-CHP group was used as the positive control. And immunohistochemistry showed high expression of BCL-2, so in addition to the first-line combination regimen, the positive control also designed a venetoclax and R-CHOP combination group. When the average tumor volume is 200mm 3 Around 6:00 p.m., patients were divided into five groups for treatment. These included a control group, a PM 5 mg / kg group, a PM 5 mg / kg group combined with venetoclax and RTX, a positive control group (Pola + R-CHP), and a venetoclax and R-CHOP group. Specific dosing schedules are shown in Tables 9 and 10.

[0247] The results are shown in Figure 30A. During the treatment process, all treatment groups significantly inhibited tumor growth in the model mice. Notably, on day 7 of administration, the tumors of 2, 3, and 1 of 5 mice in the PM 5 mg / kg, PM 5 mg / kg + Venetoclax + RTX, and Pola + R-CHOP groups, respectively, completely regressed. The PM alone and combination groups significantly outperformed the positive control Pola + R-CHP group and the Venetoclax + R-CHOP treatment group in inhibiting tumor growth. After 27 consecutive days of treatment, the PM 5 mg / kg and PM 5 mg / kg + Venetoclax + RTX groups had tumor inhibition rates of 99.71% and 100%, respectively, which were superior to the Pola + R-CHP treatment group (tumor inhibition rate of 80.99%) and the Venetoclax + R-CHOP treatment group (tumor inhibition rate of 69.36%) (Figure 30C). The tumors of 4 mice (4 / 5) in the PM 5 mg / kg group completely disappeared, the tumors of all mice in the PM 5 mg / kg + Venetoclax + RTX combination group completely disappeared, and the tumors of 2 mice (2 / 5) in the Pola + R-CHP treatment group completely disappeared, while no tumor regression occurred in the Venetoclax + R-CHOP treatment group (Figure 30D). In addition, we found an interesting phenomenon that when grouping, one mouse in each group had a large tumor mass with a volume of 300-550 mm 3 , PM alone was still effective against large tumor masses, but Pola combined with R-CHP was ineffective against large tumor masses. During treatment, mice in the PM alone group maintained normal activity, with no weight loss or other toxic side effects observed. However, mice in the other treatment groups experienced weight loss in the later stages of treatment due to long-term treatment (Figure 30B). Therefore, these results demonstrate that PM alone exhibits excellent in vivo antitumor activity in PDX model mice harboring TP53 (R248Q) and concurrent KMT2D and PIM1 mutations, surpassing the first-line combination regimens of Pola + R-CHP and Venetoclax + R-CHOP.

[0248] 6.3 PM treatment significantly inhibited tumor growth in TP53 (17P- and R175H-deficient) and mutated DLBCL (non-GCB) PDX models, with superior efficacy to BEBT-908.

[0249] Further investigations were conducted to explore the in vivo efficacy of PM in the LY-24-0398 DLBCL PDX model mice, which exhibits both TP53 (17P- and R175H) deletion and mutation, a more malignant form. Furthermore, NSG analysis of the patient's tumor tissue also detected mutations in multiple other genes associated with poor prognosis in DLBCL, including KMT2D, CARD11, BTG2, ATM, RAG1, CIITA, TNFRSF14, NOTCH3, BCL2, TET2, FGFR3, and CCND3.

[0250] When the tumor volume of the model mice reached 100-200 mm 3 Patients were divided into six groups for dosing, including control, PM 5 mg / kg, PM 10 mg / kg, PM 5 mg / kg + R-CHP, and Pola 2 mg / kg + R-CHP. Furthermore, a 50 mg / kg treatment group received BEBT-908, a dual-target HDAC and PI3K inhibitor with promising efficacy in R / R DLBCL clinical studies. Specific dosing schedules are shown in Table 8.

[0251] The results are shown in Figure 31A. Compared with the control group, the BEBT-908 50 mg / kg treatment group had no significant therapeutic effect, while the other treatment groups significantly inhibited tumor volume growth. Among them, the PM 5 mg / kg + R-CHP and Pola 2 mg / kg + R-CHP groups had the best efficacy, with tumors gradually shrinking during treatment. After 20 days of treatment, the mice were sacrificed and the tumors in each treatment group were weighed.

[0252] Tumor inhibition rates in the PM 5 mg / kg group, PM 10 mg / kg group, PM 5 mg / kg + R-CHP group, Pola 2 mg / kg + R-CHP group, and BEBT-908 50 mg / kg group were 77.66%, 87.21%, 99.85%, 99.87%, and 30.26%, respectively. No complete tumor regression was observed in the PM alone group, but complete tumor regression was observed in two mice in the PM 5 mg / kg + R-CHP group and complete tumor disappearance in three mice in the Pola + R-CHP group. Compared with the PM alone group, tumor volume was significantly reduced in the PM 5 mg / kg + R-CHP group, demonstrating that the combination of PM and R-CHP exhibited superior efficacy to PM alone (Figures 31C and D). In addition, there was no statistical difference in tumor volume and tumor weight between PM 5mg / kg+R-CHP and Pola 2mg / kg+R-CHP, and there was no significant difference in tumor inhibition rate between the two (99.85% vs 99.87%). During the treatment process, there was no significant weight loss in all treatment groups, and no other significant toxic side effects were observed (Figure 31B). The above results show that in PDX models with more malignant TP53 (17P- and R175H) deletions and mutations and combined mutations of multiple genes, PM alone also showed good anti-tumor activity, and PM combined with R-CHP had an efficacy comparable to the newly approved first-line treatment regimen Pola combined with R-CHP.

[0253] 6.4 PM is significantly more effective than the first-line regimen R-CHOP in the TP53-mutant DLBCL (ABC) CDX model

[0254] In this study, a xenograft mouse model was established by subcutaneous injection of U2932 cells to explore the efficacy of PM in TP53 mutant and dual-expression mice. 3 At around 37 h, the patients were divided into groups for treatment, including the control group, PM 5 mg / kg group, PM 10 mg / kg group, PM 5 mg / kg + RTX 10 mg / kg group, R-CHOP group, and PM 5 mg / kg + R-CHOP group. The specific dosing schedule is shown in Table 12.

[0255] The results, as shown in Figure 32A, show that compared to the control group, all treatment groups significantly inhibited tumor growth in the model mice. PM alone and in combination with RTX or R-CHOP were more effective than R-CHOP. After 16 days of treatment, mice were sacrificed and tumors in each treatment group were weighed. Tumor inhibition rates were 89.62%, 90.44%, 91.51%, and 95.45% in the PM 5 mg / kg group, PM 10 mg / kg group, PM 5 mg / kg + RTX 10 mg / kg group, and PM 5 mg / kg + R-CHOP groups, respectively, compared to 52.15% in the R-CHOP group (Figures 32C and D). In one mouse (1 / 6) treated with PM 5 mg / kg, the tumor completely disappeared. During treatment, no mice in any treatment group showed significant weight loss, and no other significant toxic side effects were observed (Figure 32B). The above results show that PM alone still has a good therapeutic effect in the treatment of TP53 mutation mouse models, and its therapeutic effect is better than the first-line treatment regimen R-CHOP.

[0256] 6.5 PM inhibits tumor growth in a PDX model of TP53 (P300L)-mutated and dual-expressing DLBCL (GCB), with significantly better efficacy than chidamide.

[0257] A TP53 mutation- and dual-expression PDX model (LY-24-0236) was established to further explore the in vivo efficacy of PM in TP53 mutation- and dual-expression mice. NGS analysis of tumor tissues from this model mouse model revealed a P300L mutation in TP53, along with mutations in CCND3, PCLO, HIST1H1C, TNFRSF14, PRDM1, TMSB4X, LYN, and SOCS1. Immunohistochemistry information: CD20(+), CD79α(+), Bcl-6(+), Bcl-2(90%+), c-myc(40%+), MUM-1(+), CD19(+), CD30(scattered+), Ki67(70%+), CD22(-), CD3(-), CD5(-), CD10(-), Cyclin D1(-), CD21(-), CD23(-); in situ hybridization EBER(-).

[0258] In LY-24-0236PDX model mice, when the average tumor volume reached 400 mm 3 Around 3 pm, group treatment began, including control group, PM 5 mg / kg group, PM 10 mg / kg group, Chi 12.5 mg / kg group, PM 5 mg / kg + R-CHP group and Pola + R-CHOP group. The specific dosing schedule is shown in Table 11.

[0259] The results, as shown in Figure 33A, showed that, with the exception of the 12.5 mg / kg chidamide group, which showed no significant therapeutic effect, all other treatment groups significantly inhibited tumor growth in the model mice. After two weeks of treatment, treatment was terminated and tumors in each treatment group were weighed. The tumor inhibition rates for the 5 mg / kg PM group, the 10 mg / kg PM group, the 12.5 mg / kg Chi group, the 5 mg / kg PM + R-CHP group, and the Pola + R-CHOP group were 48.79%, 54.94%, 29.84%, 95.65%, and 99.58%, respectively, indicating that PM alone was still effective in this model, while chidamide alone was ineffective. Furthermore, the PM combined with R-CHP group exhibited even greater antitumor activity (Figures 33C and D). During treatment, mice in the R-CHP group experienced some weight loss, while the other treatment groups showed no significant abnormalities (Figure 33B). These results further demonstrate that PM is effective in model mice harboring multiple gene mutations and dual expression, including TP53 mutations.

[0260] 6.6 PM monotherapy demonstrated excellent efficacy and a favorable safety profile in a Phase IIa clinical trial of patients with R / R DLBCL

[0261] In a phase IIa, open-label, multicenter study of PM in R / R DLBCL (NCT05563844), 30 patients were screened between November 2022 and October 2023 and randomized to PM 8.4 mg / m 2 and 11.2 mg / m 2 Dose group. Administered on days 1, 4, 8, and 11, with a 21-day treatment cycle. The median age of patients was 61 years (range, 21-77 years), and 46.7% of patients had an IPI score ≥ 3. As of the data cutoff on July 28, 2024, among the 29 evaluable patients, the ORR was 69.0% (20 / 29, 95% CI 49.2%-84.7%), including 6 CRs and 14 PRs. 8.4 mg / m 2 Of the 15 patients in the 11.2 mg / m2 dose group, 10 achieved remission, with an ORR of 66.7% (95% CI 38.4%-88.2%), including 1 CR and 9 PRs. 2 Of the 14 patients in the dose group, 10 achieved remission, with an ORR of 71.4% (95% CI 41.9%-91.6%), including 5 CRs and 5 PRs (Figures 34A and B). Five patients continued to receive treatment, with the longest treatment lasting 23 cycles (Figure 34A). The median PFS was 6.0 months. PM in the 11.2 mg / m2 dose group achieved a high CR of 35.7%. At 11.2 mg / m 2Across the dose cohorts, except for one patient who achieved a complete response (CR) and switched to other treatments, several others are still receiving treatment. Meanwhile, NGS results for available tumor tissue were available for 27 patients, and high-frequency mutations in KMT2D, CD79B, TP53, PIM1, and MYD88 were found in the PM-treated r / r DLBCL cohort. Three of the seven patients with DEL achieved a response, for an ORR of 42.9%. Of the 11 patients with TP53 mutations detected by FISH or NGS, five achieved a response, for an ORR of 45.5%. Of the 15 patients without DEL and TP53 abnormalities, 13 achieved a response, for an ORR of 86.7%. The major treatment-related adverse events (TRAEs) were similar to those of other HDACs. The most commonly reported grade ≥ 3 treatment-related events were hematologic toxicities, including thrombocytopenia (90.0%), leukopenia (70.0%), neutropenia (63.3%), lymphopenia (43.3%), and anemia (20%). Only one case of grade 3 QT prolongation was observed (3.3%). Completed and ongoing clinical trials have shown significant advantages over single-agent clinical trials of other HDAC-targeting small molecule drugs, with a favorable safety profile, fully demonstrating the therapeutic potential of PM in the treatment of relapsed or refractory diffuse large B-cell lymphoma.

Claims

1. Use of prilocastat mesylate and its analogs in the preparation of drugs for preventing and / or treating diffuse large B-cell lymphoma.

2. The use according to claim 1, characterized in that: The diffuse large B-cell lymphoma is selected from at least one of GCB subtype, ABC subtype, non-GCB subtype or other unclassified subtypes.

3. The use according to claim 2, characterized in that: The GCB subtype, ABC subtype, non-GCB subtype or other unclassified types all include subtype A and / or subtype B; the subtype A includes DLBCL subtypes in which one or more genes undergo at least one gene mutation, deletion, translocation, fusion, rearrangement and / or insertion; the subtype B includes at least one subtype of DLBCL NOS or DEL.

4. The use according to claim 3, characterized in that: In subtype A, the gene that undergoes gene mutation, deletion, translocation, fusion, rearrangement and / or insertion is selected from at least one of MYD88 gene, CD79B gene, BCL-6 gene, NOTCH2 gene, NOTCH1 gene, EZH2 gene, BCL-2 gene, MYC gene, TP53 gene, TET2 gene, SGK1 gene, CREBBP gene, MEF2B gene, KMT2C gene, KMT2D gene, EP300 gene, IRF4 gene, HIST1H1E gene, ARID1A gene, DNA methylation gene TET2, PIM1 gene, KMT2D gene, CARD11 gene, BTG2 gene, ATM gene, RAG1 gene, CIITA gene, TNFRSF14 gene, NOTCH3 gene, FGFR3 gene, CCND3 gene, PCLO gene, HIST1H1C gene, PRDM1 gene, TMSB4X gene, LYN gene or SOCS1 gene.

5. The use according to claim 4, characterized in that: The diffuse large B-cell lymphoma is selected from at least one of MCD subtype, BN2 subtype, N1 subtype, EZB-Myc positive / negative subtype, A53 subtype, ST2 subtype, DHL subtype and THL subtype.

6. The use according to any one of claims 1 to 5, characterized in that: The diffuse large B-cell lymphoma is at least one selected from DLBCL NOS, DHL, DEL lymphoma, TP53 mutation and / or deletion, TP53 mutation and / or deletion with dual expression, TP53 mutation and / or deletion with DHL, TP53 mutation and / or deletion with poor prognosis-associated gene mutation, or TP53 mutation and / or deletion with dual expression with poor prognosis-associated gene mutation; wherein the poor prognosis-associated gene is at least one selected from c-MYC, MYD88, BCL-2, CD79B, KMT2D, CARD11, BTG2, ATM, RAG1, CIITA, TNFRSF14, NOTCH3, TET2, FGFR3, CCND3, KMT2D, PIM1, PCLO, HIST1H1C, PRDM1, TMSB4X, LYN or SOCS1; Preferably, the diffuse large B-cell lymphoma is selected from TP53 mutation and BCL-2 rearrangement, ABC subtype (U2932 cell line); TP53 mutation and BCL-2 rearrangement, GCB subtype (SU-DHL-4, SU-DHL-6, Karpas 422 cell lines); c-MYC and BCL-2 rearrangement, GCB subtype (DOHH2 cell line); TP53 mutation, c-MYC and BCL-2 rearrangement, GCB subtype (OCI-LY18 cell line); TP53, MYD88 and CD79B mutation, ABC subtype (HBL-1 cell line); DLBCL NOS, GCB subtype (LY-24-0206 PDX model); DEL, non-GCB subtype (LY-24-0179 PDX model); DHL, GCB subtype (LY-24-0019 PDX models); TP53 (R248Q), KMT2D, and PIM1 mutations, non-GCB subtype (LY-24-0004 PDX model); TP53 (17P- and R175H) deletion and mutation combined with KMT2D, CARD11, BTG2, ATM, RAG1, CIITA, TNFRSF14, NOTCH3, BCL2, TET2, FGFR3, and CCND3 gene mutations, non-GCB subtype (LY-24-0398 PDX model); TP53 mutation with dual expression, ABC subtype (xenograft mouse model established with U2932 cells); or TP53 (P300L) mutation with dual expression, GCB subtype combined with CCND3, PCLO, HIST1H1C, TNFRSF14, PRDM1, TMSB4X, LYN, and SOCS1 gene mutations (LY-24-0236 DLBCL PDX model).

7. A combination drug of prilocastat mesylate and its analogs for preventing and treating diffuse large B-cell lymphoma, characterized in that: The combined drug is prilocastat mesylate and its analogs and other anti-tumor drugs administered separately or simultaneously.

8. The combined drug according to claim 7, characterized in that: The other anti-tumor drugs are selected from at least one of R-CHOP, Pola-R-CHP, R-CHP, ibrutinib, venetoclax, bendamustine, rituximab (RTX), tafasitamab, vepotuzumab (Pola), lenalidomide or epcoritamab; preferably, the other anti-tumor drugs are selected from at least one of RTX, R-CHOP, venetoclax or R-CHP.

9. The combined drug according to claim 8, characterized in that: The combination drug is composed of prilocastat mesylate and its analogs and rituximab as main drugs; preferably, the mass ratio of prilocastat mesylate and its analogs to rituximab is (1-2):(1-2); most preferably, the mass ratio of the main drugs is 1:

2.

10. The combined drug according to claim 8, characterized in that: The combination drug is composed of prilocastat mesylate and its analogs and R-CHOP as main drugs; wherein, the R-CHOP is composed of rituximab, cyclophosphamide, daunorubicin, vincristine, and prednisolone in a mass ratio of 10:20:1.25:0.2:0.15; preferably, the mass ratio of the main drugs of prilocastat mesylate and its analogs to R-CHOP is (1-2):(6-8); most preferably, the mass ratio of the main drugs is 1:6.

32.

11. The combined drug according to claim 8, characterized in that: The combination drug is composed of prilocastat mesylate and its analogs and rituximab and venetoclax as the main drugs respectively; preferably, the mass ratio of prilocastat mesylate and its analogs and rituximab and venetoclax as the main drugs is (1-2):(1-2):(6-10); most preferably, the mass ratio of the main drugs is 1:2:

8.

12. The combined drug according to claim 8, characterized in that: The combination drug is composed of prilocastat mesylate and its analogs and R-CHP as main drugs; wherein, the R-CHP is composed of rituximab, cyclophosphamide, doxorubicin, and prednisolone in a mass ratio of 10:20:1.25:0.15; preferably, the mass ratio of the main drugs of prilocastat mesylate and its analogs to R-CHP is (1-2):(6-8); most preferably, the mass ratio of the main drugs is 1:6.

28.

13. Use of the combination drug according to any one of claims 7 to 12 in the prevention and / or treatment of diffuse large B-cell lymphoma.

14. The use according to claim 13, characterized in that: The diffuse large B-cell lymphoma is selected from at least one of GCB subtype, ABC subtype, non-GCB subtype or other unclassified subtypes.

15. The use according to claim 14, characterized in that: The GCB subtype, ABC subtype, non-GCB subtype or other unclassified types all include subtype A and / or subtype B; the subtype A includes DLBCL subtypes in which one or more genes undergo at least one gene mutation, deletion, translocation, fusion, rearrangement and / or insertion; the subtype B includes at least one subtype of DLBCL NOS or DEL.

16. The use according to claim 15, characterized in that: In subtype A, the gene that undergoes gene mutation, deletion, translocation, fusion, rearrangement and / or insertion is selected from at least one of MYD88 gene, CD79B gene, BCL-6 gene, NOTCH2 gene, NOTCH1 gene, EZH2 gene, BCL-2 gene, MYC gene, TP53 gene, TET2 gene, SGK1 gene, CREBBP gene, MEF2B gene, KMT2C gene, KMT2D gene, EP300 gene, IRF4 gene, HIST1H1E gene, ARID1A gene, DNA methylation gene TET2, PIM1 gene, KMT2D gene, CARD11 gene, BTG2 gene, ATM gene, RAG1 gene, CIITA gene, TNFRSF14 gene, NOTCH3 gene, FGFR3 gene, CCND3 gene, PCLO gene, HIST1H1C gene, PRDM1 gene, TMSB4X gene, LYN gene or SOCS1 gene.

17. The use according to claim 16, characterized in that: The diffuse large B-cell lymphoma is selected from at least one of MCD subtype, BN2 subtype, N1 subtype, EZB-Myc positive / negative subtype, A53 subtype, ST2 subtype, DHL subtype and THL subtype.

18. The use according to any one of claims 13 to 17, characterized in that: The diffuse large B-cell lymphoma is at least one selected from DLBCL subtypes including DLBCL NOS, DHL, DEL lymphoma, TP53 mutation and / or deletion, TP53 mutation and / or deletion with dual expression, TP53 mutation and / or deletion with DHL, TP53 mutation and / or deletion with poor prognosis-associated gene mutation, or TP53 mutation and / or deletion with dual expression with poor prognosis-associated gene mutation; wherein the poor prognosis-associated genes include at least one of c-MYC, MYD88, BCL-2, CD79B, KMT2D, CARD11, BTG2, ATM, RAG1, CIITA, TNFRSF14, NOTCH3, TET2, FGFR3, CCND3, KMT2D, PIM1, PCLO, HIST1H1C, PRDM1, TMSB4X, LYN, or SOCS1; Preferably, the diffuse large B-cell lymphoma is selected from the group consisting of TP53 mutation and BCL-2 rearrangement, ABC subtype (U2932); TP53 mutation and BCL-2 rearrangement, GCB subtype (SU-DHL-4, SU-DHL-6, Karpas 422); c-MYC and BCL-2 rearrangement, GCB subtype (DOHH2); TP53 mutation, c-MYC and BCL-2 rearrangement, GCB subtype (OCI-LY18); TP53, MYD88 and CD79B mutation, ABC subtype (HBL-1); DLBCL NOS, GCB subtype (LY-24-0206 PDX model); DEL, non-GCB subtype (LY-24-0179 PDX model); DHL, GCB subtype (LY-24-0019 PDX models); TP53 (R248Q), KMT2D, and PIM1 mutations, non-GCB subtype (LY-24-0004 PDX model); TP53 (17P- and R175H) deletion and mutation combined with KMT2D, CARD11, BTG2, ATM, RAG1, CIITA, TNFRSF14, NOTCH3, BCL2, TET2, FGFR3, and CCND3 gene mutations, non-GCB subtype (LY-24-0398 PDX model); TP53 mutation with dual expression, ABC subtype (xenograft mouse model established with U2932 cells); or TP53 (P300L) mutation with dual expression, GCB subtype combined with CCND3, PCLO, HIST1H1C, TNFRSF14, PRDM1, TMSB4X, LYN, and SOCS1 gene mutations (LY-24-0236 DLBCL PDX model).

19. The use according to any one of claims 1 to 6, the combined drug according to any one of claims 7 to 12, or the combined drug use according to any one of claims 13 to 18, characterized in that: The said prilocastat mesylate and its analogs, or the said combination drug is a preparation prepared with prilocastat mesylate and its analogs, or a combination drug of prilocastat mesylate and its analogs and other anti-tumor drugs as the active ingredient, and with the addition of pharmaceutically acceptable excipients or auxiliary ingredients; preferably, the said preparation is an injectable preparation; more preferably, the injection route is at least one of intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intradermal injection or intramyocardial injection.

20. The use according to any one of claims 1 to 6 and 19, the combined drug according to any one of claims 7 to 12 and 19, or the combined drug use according to any one of claims 13 to 19, characterized in that: The prilocastat mesylate and its analogs are selected from compounds having the following structures: Preferably, the prilocastat mesylate and its analogs are selected from compounds having the following structures: More preferably, the prilocastat mesylate and its analogs are prilocastat mesylate for injection; More preferably, the clinical dose of Prilostat mesylate for injection is 5 mg / m 2 ~18 mg / m 2 ; Most preferably, the clinical dose of Prilostat mesylate for injection is 8.4 mg / m 2 ~11.2 mg / m 2 .

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